Tooth Whitening Compositions and Methods

ABSTRACT

Non-cytotoxic compositions for tooth whitening are disclosed. Methods of their use are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit pursuant to 35 U.S.C. § 119(e) ofU.S. Provisional Application Nos. 61/135,011, filed on Jul. 15, 2008;61/106,026, filed Oct. 16, 2008; and 61/150,685, filed Feb. 6, 2009,each of which is hereby incorporated by reference in its entiretyherein.

BACKGROUND

Efforts to whiten teeth have a long history, thought to date back to theAncient Egyptians. Modem science has provided a detailed understandingof the factors that contribute to tooth color, which has enabledimproved products and methods for whitening. The normal shade of teethis determined by the natural off-white tints of the enamel and thedentin beneath. Extrinsic and intrinsic staining also contribute totooth color.

Extrinsic staining refers to surface stains, such as those caused bytea, coffee, red wine, and other foods rich in polyphones. Extrinsicstaining primarily occurs as a result of charged surface interactionsbetween the positively-charged food molecules and the negatively-chargedtooth pellicle, the protein film on the tooth surface that is derivedfrom salivary proteins. Extrinsic stains are removed through the use ofsurfactants and/or abrasives, which cause their physical removal fromthe tooth surface.

Intrinsic staining refers to stains that exist below enamel surface, orthat penetrate below enamel surface. Intrinsic staining can happen whenfood molecules seep into enamel flaws and cracks, or, in some cases,between enamel rods. Intrinsic discoloration can also occur following achange to the structural composition or thickness of the dental hardtissues. Certain metabolic diseases and tooth trauma can also causeintrinsic staining. Tetracyline also causes intrinsic staining.

Removal of intrinsic staining is more difficult and time consuming thanremoval of extrinsic staining. Intrinsic stain removal can be achievedby a variety of methods including use of peroxides or peroxide analogs,with or without chemical, light or heat activation, to bleach thestains. This method oxidizes organic compounds within the enamel anddentin, thereby changing colored materials to non-colored materials; itdoes not remove the stain itself. Acids and dehydration methods, whichlead to opacification of enamel to obscure the subsurface stains, arealso used to remove or mask intrinsic staining.

Tooth whitening products are available over-the-counter and asprofessional services in a dentist's office. Over-the-counter productstypically contain carbamide peroxide or hydrogen peroxide as thebleaching agent. These products have concentrations of up to 21%carbamide peroxide (equivalent to 7% hydrogen peroxide) or as much as10% hydrogen peroxide. They also contain carbomers (for thickening andcontrol) and acidifiers (for peroxide stabilization in aqueoussolution), or alternatively have an anhydrous glycerin base. In-officetreatments generally use hydrogen peroxide as the oxidizer, atconcentrations of 15% or more, and typically in the 25 to 35% range. Atthese high concentrations, rubber dams, or liquid dams with propersuction, must be used to prevent gingival irritation and ingestion.Additionally, due to their high strengths, products for professionaltreatment require more thickener and more acidification to make themstable, compared to home-use products. Furthermore, professionalchair-side formulas have a secondary and often even tertiary andquaternary activators to stimulate a more rapid result. These activatorstake the form of pH modifiers, light sources, and heat sources.

Tooth whitening products, both over-the counter and particularlyprofessional treatments, have several unpleasant side effects, includingtooth sensitivity, soft tissue irritation and tooth surface changes.

Transient tooth sensitivity is the most common side effect reported.Hydrogen peroxide and carbamide peroxide have not been found to inducepathological pulpal changes in testing, although 10% carbamide peroxidehas been reported as causing mild, reversible histological changes. Itis believed that the hypersensitivity associated with whitening iscaused by dehydration, due to the acidified and thickened, substantiallyanhydrous, hydrophillic gels used in the peroxide formulations and thatare held against the teeth. Dehydration results in a negative osmoticpressure and in odontoblastic processes being drawn into the dentinaltubules. Other factors that may contribute to dehydration includewhitening lights used in in-office treatments. While sensitivity may betransient, it is a very undesirable side effect.

Oral mucosa irritation is the second most-common side effect reported.Systems using higher concentrations of hydrogen peroxide or carbamideperoxide result in more gingival irritation than lower concentrationformulations. While peroxide is regarded as safe at low concentrations,peroxide has the potential to induce cell changes at high concentrationsover an extended period of time.

Tooth surface changes have been observed for hydrogen peroxide andcarbamide peroxide in in-vitro testing. Although recent reports on thisissue have conflicting results, this aspect of tooth whitening productscurrently available remains a point of concern. In addition, toothwhitening formulations are usually acidic; acids can decalcify and etchteeth, causing a temporary opacification of underlying discolorations.These side effects often result in the need for remineralizationtherapies in connection with tooth whitening treatment, particularlythose using professional products.

Another problem with current tooth whitening formulations is rebound.Rebound is the phenomenon wherein stains re-appear after a relativelyshort period of time after tooth whitening. The time that elapsespost-treatment before this occurs varies from a few days to weeks, whileother studies claim up to 47 months before any rebound effect occurs(Leonard et al., 2001, J. Esthet. Restor. Dent. 13(6): 357-369). Onestudy found a rebound in 40% of patients at six months with use ofconcentrations ranging from 16%-18% carbamide peroxide (Brunton et al.,2004, Oper. Dent. 29(6): 623-626).

Chlorine dioxide (ClO₂) has been suggested as an alternative to peroxidebased bleaching agents for tooth whitening applications. Chlorinedioxide (ClO₂) is well known as a disinfectant as well as a strongoxidizing agent. The bactericidal, algaecidal, fungicidal, bleaching,and deodorizing properties of chlorine dioxide are also well known.

Chlorine dioxide (ClO₂) is a neutral compound of chlorine in the +IVoxidation state. It disinfects by oxidation; however, it does notchlorinate. It is a relatively small, volatile, and highly energeticmolecule, and a free radical even in dilute aqueous solutions. Chlorinedioxide functions as a highly selective oxidant due to its one-electrontransfer mechanism in which it is reduced to chlorite (ClO₂ ⁻). The pKafor the chlorite ion/chlorous acid equilibrium, is extremely low at pH1.8. This is remarkably different from the hypochlorousacid/hypochlorite base ion pair equilibrium found near neutrality, andindicates that the chlorite ion will exist as the dominant species indrinking water.

One of the most important physical properties of chlorine dioxide is itshigh solubility in water, particularly in chilled water. In contrast tothe hydrolysis of chlorine gas in water, chlorine dioxide in water doesnot hydrolyze to any appreciable extent, but remains in solution as adissolved gas.

The traditional method for preparing chlorine dioxide involves reactingsodium chlorite with gaseous chlorine (Cl₂(g)), hypochlorous acid(HOCl), or hydrochloric acid (HCl). The reactions are:

2NaClO₂+Cl₂(g)=2ClO₂(g)+2NaCl   [1a]

2NaClO₂+HOCl=2ClO₂(g)+NaCl+NaOH   [1b]

5NaClO₂+4HCl=4ClO₂(g)+5NaCl+2H₂O   [1c]

-   Reactions [1a] and [1b] proceed at much greater rates in acidic    medium, so substantially all traditional chlorine dioxide generation    chemistry results in an acidic product solution having a pH below    3.5. Also, because the kinetics of chlorine dioxide formation are    high order in chlorite anion concentration, chlorine dioxide    generation is generally done at high concentration (>1000 ppm),    which must be diluted to the use concentration for application.

Chlorine dioxide may also be prepared from chlorate anion by eitheracidification or a combination of acidification and reduction. Examplesof such reactions include:

2NaClO₃+4HCl→2ClO₂+Cl₂+2H₂O+2NaCl   [2a]

2HClO₃+H₂C₂O₄→2ClO₂+2CO₂+2H₂O [2b]

2NaClO₃+H₂SO₄+SO₂→2ClO₂+2NaHSO₄   [2c]

-   At ambient conditions, all reactions require strongly acidic    conditions; most commonly in the range of 7-9 N. Heating of the    reagents to higher temperature and continuous removal of chlorine    dioxide from the product solution can reduce the acidity needed to    less than 1 N.

A method of preparing chlorine dioxide in situ uses a solution referredto as “stabilized chlorine dioxide.” Stabilized chlorine dioxidesolutions contain little or no chlorine dioxide, but rather, consistsubstantially of sodium chlorite at neutral or slightly alkaline pH.Addition of an acid to the sodium chlorite solution activates the sodiumchlorite, and chlorine dioxide is generated in situ in the solution. Theresulting solution is acidic. Typically, the extent of sodium chloriteconversion to chlorine dioxide is low and a substantial quantity ofsodium chlorite remains in the solution.

U.S. Pat. No. 6,582,682 discloses an oral care composition comprising“stabilized chlorine dioxide” that, upon exposure to the mildly acidicpH in the oral cavity, produces chlorine dioxide.

U.S. Pat. No. 6,479,037 discloses preparing a chlorine dioxidecomposition for tooth whitening wherein the composition is prepared bycombining a chlorine dioxide precursor (CDP) portion with an acidulant(ACD) portion. The CDP portion is a solution of metal chlorite at a pHgreater than 7. The ACD is acidic, preferably having a pH of 3.0 to 4.5.The CDP is applied to the tooth surface. The ACD is then applied overthe CDP to activate the metal chlorite and produce chlorine dioxide. ThepH at the contact interface is preferably less than 6 and, mostpreferably, in the range of about 3.0 to 4.5. Thus, the resultingchlorine dioxide composition on the tooth surface is acidic.Additionally, this method exposes the oral mucosa to possible contactwith a strongly highly acidic reagent (ACD).

There is a need in the art for tooth whitening compositions and methodswith reduced side effects.

SUMMARY

The following embodiments meet and address these needs. The followingsummary is not an extensive overview. It is intended to neither identifykey or critical elements of the various embodiments, not delineate thescope of them.

In one aspect, a method of whitening a tooth surface is provided. Themethod comprises comprising contacting the surface of a tooth with anefficacious amount of a substantially non-irritating composition,wherein the composition comprises a bleaching agent that whitens thecontacted tooth surface, wherein the bleaching agent is chlorinedioxide. In some embodiments, the non-irritating composition issubstantially non-cytotoxic. In some embodiments, the substantiallynon-irritating composition comprises less than about 0.2 milligramsoxy-chlorine anion per gram composition.

In an embodiment, the substantially non-irritating composition comprisesabout 5 to about 1000 ppm chlorine dioxide. In another embodiment, thesubstantially non-irritating composition comprises about 30 to about 40ppm chlorine dioxide. In some embodiments, the substantiallynon-irritating composition has a pH from about 4.5 to about 11. In otherembodiments, the non-irritating composition has a pH from about 5 toabout 9, or a pH greater than about 6 and less than about 8.

The substantially non-irritating composition can be a thickened fluidcomposition comprising a thickener component in some embodiments. Insome embodiments of the thickened fluid composition, the thickenercomponent is selected from the group consisting of naturalhydrocolloids, semisynthetic hydrocolloids, synthetic hydrocolloids, andclay. In an embodiment, the thickener component is a semisynthetichydrocolloid. An exemplary semisynthetic hydrocolloid iscarboxymethylcellulose, such as sodium carboxymethylcellulose.

In some embodiments, contacting the tooth with the non-irritatingcomposition does not: substantially damage hard tooth tissue;substantially reduce enamel microhardness; substantially reduce dentinmicrohardness; cause tooth sensitivity and/or substantially increasesurface roughness of at least one of enamel and dentin. In someembodiments, the rate and/or extent of rebound is reduced compared to aperoxide bleaching agent.

In some embodiments, the non-irritating composition contacts soft oraltissue. In some embodiments, no protection of the gums is required.Optionally, in some embodiments, the non-irritating composition is anoral rinse.

In some embodiments, the contacted tooth surface is whitened by at leastabout 3 shade value units. In other embodiments, the contacted toothsurface is whitened by at least about 6 shade value units.

In another aspect, a method of whitening a tooth surface is provided,comprising the step of contacting the surface of a tooth with anefficacious amount of a composition that does not substantially damagehard tooth tissue, wherein the composition comprises a bleaching agentthat whitens the contacted tooth surface, wherein the bleaching agent ischlorine dioxide. In some embodiments, the composition is substantiallynon-cytotoxic and/or substantially non-irritating. In some embodiments,the composition comprises less than about 0.2 milligrams oxy-chlorineanion per gram composition.

In an embodiment, the composition comprises about 5 to about 1000 ppmchlorine dioxide. In another embodiment, the composition comprises about30 to about 40 ppm chlorine dioxide. In some embodiments, thecomposition has a pH from about 4.5 to about 11. In other embodiments,the composition has a pH from about 5 to about 9, or a pH greater thanabout 6 and less than about 8.

The composition can be a thickened fluid composition comprising athickener component in some embodiments. In some embodiments of thethickened fluid composition, the thickener component is selected fromthe group consisting of natural hydrocolloids, semisynthetichydrocolloids, synthetic hydrocolloids, and clay. In an embodiment, thethickener component is a semisynthetic hydrocolloid. An exemplarysemisynthetic hydrocolloid is carboxymethylcellulose, such as sodiumcarboxymethylcellulose.

In some embodiments, contacting the tooth with the composition does not:substantially reduce enamel microhardness; substantially reduce dentinmicrohardness; cause tooth sensitivity; and/or substantially increasesurface roughness of at least one of enamel and dentin. In someembodiments, the rate and/or extent of rebound is reduced compared to aperoxide bleaching agent.

In some embodiments, the composition contacts soft oral tissue. In someembodiments, no protection of the gums is required. Optionally, in someembodiments, the composition is an oral rinse.

In some embodiments, the contacted tooth surface is whitened by at leastabout 3 shade value units. In other embodiments, the contacted toothsurface is whitened by at least about 6 shade value units.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the various compositions and methods,there are depicted in the drawings certain embodiments of the invention.However, the compositions and their methods of use are not limited tothe precise arrangements and instrumentalities of the embodimentsdepicted in the drawings.

FIG. 1 is a bar graph depicting tooth whitening data for a non-cytotoxicClO₂-containing composition and a commercial over-the-counter (OTC)product having 10% hydrogen peroxide as a function of total treatmenttime. ClO₂=data for non-cytotoxic ClO₂-containing composition. OTC=datafor commercial product having 10% hydrogen peroxide.

FIG. 2 is a graph depicting tooth whitening data for a non-cytotoxicClO₂-containing composition in comparison to a professional whiteninggel comprising 36% hydrogen peroxide as the bleaching agent.

FIG. 3A-3C are a series of representative scanning electron microscopy(SEM) photomicrography images of enamel surface at 2500× magnification.FIG. 3A is the enamel of an untreated tooth. FIG. 3B is enamel surfaceafter treatment with a non-cytotoxic ClO₂-containing composition. FIG.3C is enamel surface after treatment with a professional whitening gelcontaining 36% hydrogen peroxide.

FIGS. 4A-4C are a series of representative SEM microphotograph images ofdentin surface at 5000× magnification. FIG. 4A is the dentin of anuntreated tooth. FIG. 4B is dentin surface after treatment with an OTCwhitening gel containing 10% hydrogen peroxide. FIG. 4C is dentinsurface after treatment with a non-cytotoxic ClO₂-containingcomposition.

DETAILED DESCRIPTION

Provided herein is a method of whitening a tooth using a compositioncomprising a bleaching agent, wherein the composition is notsubstantially cytotoxic. In a preferred embodiment, the bleaching agentcan be chlorine dioxide.

Definitions

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art. Generally, the nomenclature used herein andthe laboratory procedures in cytopathicity analysis, microbial analysis,organic and inorganic chemistry, and dental clinical research are thosewell known and commonly employed in the art.

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “about” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which it can be used.Generally, “about” encompasses a range of values that are plus/minus 10%of a reference value. For instance, “about 25%” encompasses values from22.5% to 27.5%.

It is understood that any and all whole or partial integers between anyranges set forth herein are included herein.

As used herein, “biocidal” refers to the property of inactivating orkilling pathogens, such as bacteria, algae, and fungi.

As used herein, “NaDCCA” refers to sodium dichloroisocyanurate and/orthe dihydrate thereof.

As used herein, “tooth whitening” refers to a lightening of tooth shaderelative to the tooth shade prior to treatment. Lightening can beassessed on an isolated or an in situ tooth by standard, art-recognizedmethods of assessing tooth shade, which include qualitative,quantitative and semi-quantitative methods. For instance, lightening maybe assessed by simple visual inspection, e.g., by comparing “before” and“after” photographs of the treated teeth. Alternatively, a tooth may bedeemed whitened when the tooth shade relative to the tooth shade priorto treatment is two or more shades lighter, as assessed by Vitaclassical shade guide (preferably, under controlled visible lightconditions) or two or more levels as assessed using the VitaBleachedguide 3D-MASTER Shade system, which utilizes a multiple colorspectrophotometer and includes half lightness levels. A difference ofone shade is referred to herein as a “shade value unit” (SVU). Thus, forexample, a difference of two shades is a 2 SVU difference.

“Bleaching agent” as used herein refers to the active ingredient, orcombination of ingredients, in a composition that causes the lighteningand/or removal of the chromagens that contribute to the dark shade of atooth.

As used herein, an “efficacious amount” of a bleaching agent is intendedto mean any amount of a bleaching agent that will result in toothwhitening, as defined herein, using methods of assessment known to theskilled artisan, as discussed above of a tooth, with one or moretreatments.

As used herein, “cytotoxic” refers to the property of causing lethaldamage to mammalian cell structure or function. A composition is deemed“substantially non-cytotoxic” or “not substantially cytotoxic” if thecomposition meets the United States Pharmacopeia (USP) biologicalreactivity limits of the Agar Diffusion Test of USP <87> “BiologicalReactivity, in vitro,” (approved protocol current in 2007) when theactive pharmaceutical ingredient (API) is present in an efficaciousamount.

As used herein, “irritating” refers to the property of causing a localinflammatory response, such as reddening, swelling, itching, burning, orblistering, by immediate, prolonged, or repeated contact. A compositionis deemed “substantially non-irritating” or “not substantiallyirritating” if the composition is judged to be slightly or notirritating using any standard method for assessing oral mucosalirritation. Non-limiting examples of such methods include: HET-CAM(hen's egg test-chorioallantoic membrane); slug mucosal irritation test;and in vitro tests using tissue-engineered oral mucosa.

As used herein, “hard tooth tissue” refers to at least one of enamel anddentin.

As used herein, “hard tooth tissue damage” refers to at least one of areduction of microhardness of enamel, a reduction of microhardness ofdentin, an increase in the surface roughness of enamel and an increasein the surface roughness of dentin.

As used herein, a composition “does not substantially damage hard toothtissue” if one or more of the following is met for a tooth aftertreatment relative to the tooth prior to treatment: 1) enamelmicrohardness is decreased by an amount less than about 15% and/or thereduction is not statistically significant at the 5% confidence level;2) dentin microhardness is decreased by an amount less than about 15%and/or the reduction is not statistically significant at the 5%confidence level; 3) enamel surface roughness is increased by an amountno more than about 20% and/or the increase is not statisticallysignificant at the 5% confidence level; and 4) dentin surface roughnessis increased by an amount no more than about 8% and/or the increase isnot statistically significant at the 5% confidence level.

As used herein, “oxy-chlorine anion” refers to chlorite (ClO₂ ⁻) and/orchlorate (ClO₃ ⁻) anions.

As used herein, “substantially pure chlorine dioxide solution” refers toa solution of chlorine dioxide that has a non-cytotoxic concentration ofoxy-chlorine anion. As used herein, “substantially pure chlorine dioxidesolution” also refers to a concentrated solution of chlorine dioxidethat contains a concentration of oxy-chlorine anion that upon dilutionto an efficacious amount of chlorine dioxide is not cytotoxic withrespect to the concentration of oxy-chlorine anion.

The phrase “thickened fluid composition” encompasses compositions whichcan flow under applied shear stress and which have an apparent viscositywhen flowing that is greater than the viscosity of the correspondingaqueous chlorine dioxide solution of the same concentration. Thisencompasses the full spectrum of thickened fluid compositions,including: fluids that exhibit Newtonian flow (where the ratio of shearrate to shear stress is constant and viscosity is independent of shearstress), thixotropic fluids (which require a minimum yield stress to beovercome prior to flow, and which also exhibit shear thinning withsustained shear), pseudoplastic and plastic fluids (which require aminimum yield stress to be overcome prior to flow), dilantant fluidcompositions (which increase in apparent viscosity with increasing shearrate), and other materials which can flow under applied yield stress.

A “thickener component,” as the phrase is used herein, refers to acomponent that has the property of thickening a solution or mixture towhich it is added. A “thickener component” can be used to make a“thickened fluid composition” as described above.

By “apparent viscosity” is meant the ratio of shear stress to shear rateat any set of shear conditions that result in flow. Apparent viscosityis independent of shear stress for Newtonian fluids and varies withshear rate for non-Newtonian fluid compositions.

The term “particulate” is used herein to refer to all solid materials.By way of a non-limiting example, particulates may be interspersed witheach other to contact one another in some way. These solid materialsinclude particles of any size, and combinations of particles ofdifferent sizes.

As used herein, “remineralization” refers to the process of repair ofacid damaged tooth structure by the recrystallization of mineral saltson or within the tooth architecture.

As used herein, “demineralization” refers to the process of mineral lossfrom teeth caused by acid, chelating agents or other accelerants ofdissolution. Demineralization can occur on tooth surfaces and/or belowtooth surfaces, depending on the composition of the demineralizingagent, the contacting medium, the concentration and the pH.

Description

The discovery turns at least on the realization that potent and rapidtooth whitening can be achieved using chlorine dioxide compositions thatare non-cytotoxic, cause minimal damage to hard tooth tissues and aresubstantially non-irritating to soft tissues of the mouth. As shownherein, cytotoxicity of chlorine dioxide-containing compositions resultspredominantly from the presence of oxy-chlorine anions, and not from thepresence of chlorine, which can be a product of chlorine dioxidedecomposition. Further as shown herein, substantially non-cytotoxic,non-irritating and non-hard tissue damaging compositions comprisingchlorine dioxide as a bleaching agent provide rapid and extensive toothwhitening. Indeed, the rate and extent of tooth whitening achieved bysuch compositions were found to be superior to over-the-counterperoxide-based tooth whitening compositions and comparable to certainprofessional peroxide-based tooth whitening compositions. In someembodiments, the tooth surface can be whitened by at least about 3 shadevalue units, at least about 5 shade value units, or at least about 6shade value units. Advantageously, substantially non-cytotoxicchlorine-dioxide containing compositions do not adversely affect enamelor dentin microhardness to a significant extent. Furthermore, thecompositions do not substantially increase surface roughness of teeth,even after extended contact with the composition. Thus, tooth whiteningcan be obtained without substantially damaging hard tooth tissue. It isbelieved therefore that tooth whitening in accordance with a disclosedmethod will have reduced or substantially reduced tooth sensitivity andsoft tissue irritation, compared to peroxide-based tooth whiteningproducts currently available. It is further contemplated that the extentand/or rate of rebound will be reduced, compared to peroxide-based toothwhitening products currently available.

Thus, a method of whitening a tooth is provided, wherein the methodcomprises contacting a tooth surface with an efficacious amount of acomposition, wherein the composition comprises chlorine dioxide as ableaching agent. The composition is substantially non-cytotoxic,substantially non-irritating to soft tissues in the mouth and does notsubstantially damage hard tooth tissue.

I. Composition

The substantially non-cytotoxic composition used in the practice of themethod is an aqueous fluid that comprises chlorine dioxide, or thereactants for generating chlorine dioxide, as a bleaching agent. Inpreferred embodiments, the composition comprises a thickener componentwhich renders the composition a thickened aqueous fluid. In anotherpreferred embodiment, the composition can be an oral rinse that may beheld in the mouth in contact with teeth, as well as soft tissue.

Compositions useful in the practice of the method comprise at leastabout 5 ppm chlorine dioxide, preferably at least about 20 ppm, and morepreferably at least about 30 ppm. Typically, the amount of chlorinedioxide can be up to about 1000 ppm, preferably up to about 700 ppm,more preferably up to about 500 ppm and more preferably still up toabout 200 ppm. In certain embodiments, the chlorine dioxideconcentration ranges from about 5 to about 700 ppm, preferably fromabout 20 to about 500 ppm, and most preferably from about 30 to about200 ppm chlorine dioxide. In one embodiment, the composition comprisesabout 30 to about 40 ppm chlorine dioxide. In one embodiment, thecomposition comprises about 30 ppm. In another embodiment, thecomposition comprises about 40 ppm.

Soft tissue irritation can result from highly reactive oxygen species,such as those found in peroxide based compositions. Soft tissueirritation can also result from extremes of pH, both acidic and basic.To minimize soft tissue irritation of the chlorine dioxide containingcomposition, the substantially non-cytotoxic composition has a pH of atleast about 3.5. To minimize possible hard surface erosion, thecomposition has a pH of at least about 4.5. Preferably, the compositionhas a pH of at least about 5, and more preferably still, greater thanabout about 6. In certain embodiments, the pH ranges from about 4.5 toabout 11, more preferably from about 5 to about 9, and more preferablystill, greater than about 6 and less than about 8. In one embodiment,the pH is about 6.5 to about 7.5. Irritation is not believed to resultfrom the concentration of oxy-chlorine anions.

For compositions comprising chlorine dioxide, as shown herein,cytotoxicity results predominantly from the presence of oxy-chlorineanions. Accordingly, a composition comprising chlorine dioxide thatcomprises zero milligram (mg) oxy-chlorine anion per gram composition tono more than about 0.25 mg oxy-chlorine anion per gram composition,preferably zero to about 0.24, 0.23, 0.22, 0.21, or 0.20 mg oxy-chlorineanion per gram composition, more preferably zero to about 0.19, 0.18,0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, or 0.10 mg oxy-chlorine anionper gram composition and more preferably still from zero to about 0.09,0.08, 0.07, 0.06, 0.05 or 0.04 mg oxy-chlorine anion per gramcomposition, absent other constituents that contribute to cytotoxicity,is substantially non-cytotoxic.

A substantially non-cytotoxic composition comprising chlorine dioxidecan be prepared using a substantially pure chlorine dioxide solutionhaving a neutral pH. Preferably, the solution has a pH from about 5 toabout 9, and more preferably, from about 6.5 to about 7.5. One source ofa substantially pure chlorine dioxide solution can be chlorine dioxideis prepared using an ASEPTROL (BASF Corp., Florham Park, N.J.) material,which are described in commonly-assigned U.S. Pat. Nos. 6,432,322 and6,699,404. These patents disclose solid bodies for preparing highlyconverted solutions of chlorine dioxide when added to water. The solidbody comprises a metal chlorite such as sodium chlorite, an acid sourcesuch as sodium bisulfate and optionally a source of free halogen such asthe sodium salt of dichloroisocyanuric acid or a hydrate thereof.ASEPTROL materials provide a way to efficiently generate chlorinedioxide at substantially neutral pH, thus avoiding tooth-compatibilityproblems existing with earlier, acidic chlorine dioxide based oralproducts. ASEPTROL material in an aqueous fluid has an extremely highconversion rate, resulting in high concentrations of chlorine dioxideand low concentrations of oxy-chlorine anion.

Another method of preparing substantially pure chlorine dioxide can beto prepare a chlorine dioxide source solution by any known method, thenbubbling air through that solution (sparging) and into a secondcontainer of deionized water, to prepare the product solution ofsubstantially pure chlorine dioxide. Only ClO₂ and possibly some watervapor is transferred from the source solution to the product solution.All the salt ingredients remain behind in the source solution. Thus,there are no oxy-chlorine anions in the substantially pure productsolution. While the chlorine dioxide may undergo a degree ofdecomposition, the rate is relatively slow. By keeping the solutioncapped and protected from ultraviolet exposure, the decomposition ratecan be slowed to a rate of about 5 to about 25% reduction in chlorinedioxide in 7 days. Substantially pure chlorine dioxide may also beprepared using a pervaporation technique, such as that disclosed in U.S.Pat. No. 4,683,039. In addition, a metal chlorite and an acid source canbe reacted in solution to yield high conversion to chlorine dioxide andproduce a greater than 2000 ppm chlorine dioxide solution. Theconcentrated solution can then be buffered to a neutral pH. Similarly, achlorine dioxide solution can be prepared the composition described inU.S. Pat. No. 5,399,288, which yields a high concentration chlorinedioxide solution at acidic pH. The concentrated solution can then bebuffered to achieve a substantially neutral pH to prepare asubstantially pure chlorine dioxide solution.

Oxy-chlorine anions can be measured in these solutions using any methodknown to those skilled in the art, including ion chromatographyfollowing the general procedures of EPA test method 300 (Pfaff, 1993,“Method 300.0 Determination of Inorganic Anions by Ion Chromatography,”Rev. 2.1, US Environmental Protection Agency) or a titration methodbased on an amperometric method (Amperometric Method II in Eaton et al,ed., “Standard Methods for the Examination of Water and Wastewater”19^(th) edition, American Public Health Association, Washington D.C.,1995). Alternatively, oxy-chlorine anions may be measured by a titrationtechnique equivalent to the amperometric method, but which uses theoxidation of iodide to iodine and subsequent titration with sodiumthiosulfate to a starch endpoint in place of the amperometric titration;this method is referred to herein as “pH 7 buffered titration.” Achlorite analytical standard can be prepared from technical grade solidsodium chlorite, which is generally assumed to comprise about 80% byweight of pure sodium chlorite.

To prepare a thickened aqueous composition comprising chlorine dioxidethat is substantially not cytotoxic, non-irritating and does not damagehard tooth tissue, the substantially pure chlorine dioxide solution canthen be combined with a thickener component and an aqueous medium. Thematerials and methods also encompass a two-component whitening systemcomprising a first component comprising a substantially pure chlorinedioxide solution and a second component comprising a thickener componentin an aqueous medium. Combination of the first and second componentsyields a non-cytotoxic composition comprising an amount of chlorinedioxide efficacious for tooth whitening. Chlorine dioxide in solutionwill decompose over time. To avoid problems arising from suchdecomposition, including loss of efficacy and generation of chloriteanions, the substantially pure chlorine dioxide solution can be preparedimmediately before its combination with a thickener component and anaqueous medium. In addition, the composition can be prepared immediatelybefore its use.

“Immediately before” as used herein refers to a period no greater thanthat which would result in diminished efficacy or evidence ofcytotoxicity. Generally, “immediately before” is less than about 14days, and preferably no greater than about 24 hours and more preferablyno greater than about 2 hours. Preferably, the substantially purechlorine dioxide solution is prepared within about 8 hours of thepreparation of the composition. Precautions are also taken to avoidexposing the chlorine dioxide solution or the prepared composition tostrong ultraviolet light or elevated temperature (e.g., temperaturegreater than ambient temperature, about 25° C.).

Methods of preparing substantially non-cytotoxic thickened compositionscomprising chlorine dioxide are also disclosed in commonly-assigned U.S.provisional application No. 61/150,685, filed Feb. 6, 2009, entitled“Non-Cytotoxic Chlorine Dioxide Fluids,” incorporated herein byreference in its entirety.

Methods of preparing thickened compositions comprising chlorine dioxideare also disclosed in commonly-assigned U.S. Pat. Publication Nos.2006/0169949 and 2007/0172412. In practicing the methods described inthese two publications, steps must be taken (as described herein) tocontrol the oxy-chlorine concentration so as to produce a non-cytotoxiccomposition.

A substantially non-cytotoxic composition comprising chlorine dioxidecan also be prepared using a particulate precursor of ClO₂ and anaqueous thickened fluid composition. Another aspect encompasses atwo-component whitening comprising a first component comprising aparticulate precursor of chlorine dioxide and a second componentcomprising a thickener component in an aqueous medium. Combination ofthe first and second components yields a non-cytotoxic compositioncomprising an amount of chlorine dioxide efficacious for toothwhitening. In an embodiment, the components of the system are adapted tobe admixed and applied to the teeth from a dental tray for sustainedcontact. Precursors of ClO₂ include metal chlorites, metal chlorates, anacid source and an optional halogen source. The particulate precursormay comprise one of these or any combination of these. The particulateprecursor can be an ASEPTROL product, more preferably it can be ASEPTROLS-Tab2. ASEPTROL S-Tab2 has the following chemical composition by weight(%): NaClO₂ (7%); NaHSO₄ (12%); NaDCC (1%); NaCl (40%); MgCl₂ (40%).Example 4 of U.S. Pat. No. 6,432,322 describes an exemplary manufactureprocess of S-Tab2. Granules can be produced, either by comminutingpressed S-Tab2 tablets, or by dry roller compaction of the non-pressedpowder of the S-Tab2 components, followed by breakup of the resultantcompacted ribbon or briquettes, and then screening to obtain the desiredsize granule. Upon exposure to water or an aqueous thickened fluid,chlorine dioxide can be generated from the ASEPTROL granules. In oneembodiment, a substantially non-cytotoxic composition comprisingchlorine dioxide can be prepared by combining −40 mesh granules with anaqueous thickened fluid. In one aspect, the thickener component of thethickened fluid can be carboxymethylcellulose (CMC). Preferably, theaqueous thickened fluid can be prepared sufficiently in advance ofcombining with the ASEPTROL granules to enable the complete hydration ofthe thickener component. In one embodiment, the thickened fluidcomposition is formed by adding high viscosity NaCMC powder to distilledwater. The NaCMC is allowed to hydrate for at least 8 hours, and thenthe mixture is stirred to homogenize it. A substantially non-cytotoxiccomposition for tooth whitening is then prepared by mixing the sizedASEPTROL granules with the NaCMC thickened fluid composition.

The thickened fluid composition may also be formed in situ, whereinsaliva serves as the aqueous medium. In one embodiment, a mixture ofASEPTROL granules and a thickener component can be formed into a shape,for instance by addition of a malleable wax, and the shape is thenapplied to teeth. Saliva activates the granules, forming chlorinedioxide and the thickener component hydrates, thereby forming thethickened fluid composition in situ. In another embodiment, a mixture ofASEPTROL granules and a thickener component can be placed on a dentalstrip or a dental film or in a dental tray. A dental strip refers to asubstantially planar object made of a plastic backbone that issufficiently flexible to affix to teeth. A dental film refers to asubstantially planar object made of a pliable, conformable material thatcan be substantially fitted to the surface of teeth. Optionally, thedental strip is dissolvable in an aqueous medium, such as saliva. Thestrip, film or tray can be positioned on teeth, and saliva serves as theaqueous medium as described above to produce the thickened fluidcomposition in situ. Alternatively, the mixture on the strip or tray canbe contacted with water or aqueous medium prior to positioning on theteeth.

There is no extremely accurate method for measuring oxy-chlorine aniondirectly in a thickened fluid composition. This value can be accuratelyestimated, however, by measuring the oxy-chlorine anion in the aqueoussolution (prior to thickening), and adjusting the final concentration onthe basis of weight of the final thickened fluid. The titration methoddescribed elsewhere herein is contemplated as useful in assessing boththe chlorine dioxide concentration and the oxy-chlorine anionconcentration in thickened fluid compositions. It is contemplated thatoxy-chlorine anions in a thickened fluid composition can be measuredusing ion chromatography as described elsewhere herein, provided stepsare taken to preclude fouling of the column by the hydrated thickenercomponent. One such step is the use of molecular weight filters toremove the hydrated thickener component, such as hydrated CMC, prior toapplication to the chromatography column. If necessary, the thickenedfluid composition may be diluted with water, prior to analysis, toreduce its viscosity or otherwise allow it to be more readily tested.One of skill in the art can readily determine empirically whether agiven formulation has a sufficiently low oxy-chlorine concentration bydetermining if the formulation is cytotoxic using USP biologicalreactivity limits of the Agar Diffusion Test of USP <87>.

The aqueous thickened fluid composition used in the method may compriseany thickener component in an aqueous medium, wherein the thickenedfluid composition can be non-cytotoxic and non-irritating to softtissues, in particular oral mucosa, and causes minimal damage to hardtissues, such as tooth enamel and dentin. In addition, the thickener ispreferably not adversely affected by the bleaching agent on the timescale of composition preparation and use in treatment. Many thickeneragents are known in the art, including, but not limited to carbomers(e.g., CARBOPOL thickeners, Lubrizol Corp., Wickliffe, Ohio),carboxymethylcellulose (CMC), ethylcellulose, hydroxyethylcellulose,hydroxypropyl cellulose, natural smectite clays (e.g., VEEGEM, R. T.Vanderbilt Co., Norwalk, Conn.), synthetic clays (e.g., LAPONITE(Southern Clay Products, Gonzales, Tex.), methylcellulose,superabsorbent polymers such as polyacrylates (e.g., LUQUASORB 1010,BASF, Florham Park, N.J.), poloxamers (PLURONIC, BASF, Florham Park,N.J.), polyvinyl alcohol, sodium alginate, tragacanth, and xanthan gum.Such thickening agents may be categorized into four groups: naturalhydrocolloids (also referred to as “gum”), semisynthetic hydrocolloids,synthetic hydrocolloids, and clay. Some examples of naturalhydrocolloids include acacia, tragacanth, alginic acid, carrageenan,locust bean gum, guar gum, and gelatin. Non-limiting examples ofsemisynthetic hydrocolloids include methylcellulose and sodiumcarboxymethylcellulose. Some examples of synthetic hydrocolloids (alsoreferred to as “polymers” including polymers, cross-linked polymers, andcopolymers) include polyacrylates, superabsorbent polymers, highmolecular weight polyethylene glycols and polypropylene glycols,polyethylene oxides and CARBOPOL. Non-limiting examples of clay(including swelling clay) include LAPONITE, attapulgite, bentonite andVEEGUM. An exemplary thickener component is a semisynthetichydrocolloid. Another exemplary thickener component can be a highviscosity sodium carboxymethylcellulose (NaCMC powder).

CMC is a cellulose derivative with carboxymethyl groups (—CH₂—COOH)bound to some of the hydroxyl groups of the glucopyranose monomers thatmake up the cellulose backbone. It can be synthesized by thealkali-catalyzed reaction of cellulose with chloroacetic acid. The polar(organic acid) carboxyl groups render the cellulose soluble andchemically reactive. The functional properties of CMC depend on thedegree of substitution of the cellulose structure (i.e., how many of thehydroxyl groups have taken part in the substitution reaction), and chainlength of the cellulose backbone structure.

CMC is available in a range of viscosity grades and to USP standards.High viscosity CMC, such as type CA194 from Spectrum ChemicalManufacturing Company, has a viscosity of between 1500 and 3000 cps at25° C. at 1% concentration in water.

The composition can be a fluid. In some embodiments, the fluid can be athickened fluid having flow properties suitable for applying the fluidto a tooth surface and leaving the fluid in place for the duration of atooth whitening treatment (e.g., about 5 to about 60 minutes).Accordingly, a pseudoplastic composition with a sufficient yield pointto retains its shape when applied to teeth but low enough to be readilyremoved by wiping is advantageous in practicing the method. Inembodiments where the composition is contacted to a tooth using a dentaltray, strip, or similar device, the composition should have sufficientadhesion to hold the device in place. Exemplary adhesion agents aredisclosed in U.S. Pat. Publication No. 2008/0025925.

The composition may optionally comprise other components. Suchcomponents include, but are not limited to, sweeteners, flavorants,coloring agents, and fragrances. Sweeteners include sugar alcohols.Exemplary sugar alcohols include sorbital, xylitol, lactitol, mannitol,maltilol, hydrogenated starch hydrolysate, erythritol, reducingparatinose, and mixtures thereof. Flavoring agents include, e.g.,natural or synthetic essential oils, as well as various flavoringaldehydes, esters, alcohols, and other materials. Examples of essentialoils include oils of spearmint, peppermint, wintergreen, sassafras,clove, sage, eucalyptus, marjoram, cinnamon, lemon, lime, grapefruit,and orange. Coloring agents include a colorant approved forincorporation into a food, drug, or cosmetic by a regulatory agency,such as, for example, FD&C or D&C pigments and dyes approved by the FDAfor use in the United States. Fragrances include menthol, menthylacetate, menthyl lactate, camphor, eucalyptus oil, eucalyptol, anethole,eugenol, cassia, oxanone, α-irisone, propenyl guaiethol, thymol,linalool, benzaldehyde, cinnamaldehyde, N-ethyl-p-menthan-3-carboxamine,N,2,3-trimethyl-2-isopropylbutanamide, 3-(1-menthoxy)-propane-1,2-diol,cinnamaldehyde glycerol acetal (CGA), menthone glycerol acetal (MGA),and the like.

Other optional components for the composition include: antibacterialagents (in addition to chlorine dioxide), enzymes, malodor controllingagents (in addition to chlorine dioxide), cleaning agents, such asphosphates, antigingivitis agents, antiplaque agents, antitartar agents,anticaries agents, such as a source of fluoride ion, antiperiodontitisagents, nutrients, antioxidants, and the like. Advantageously, themethod can reduce or eliminate the need for agents to relieve toothsensitivity and agents for remineralizing the enamel. However, suchagents may be included in the composition in some embodiments. Exemplaryagents are well-known in the art. See e.g., U.S. Pat. Publication Nos.2005/0287084; 2006/0263306; 2007/0259011; and 2008/0044363.

It is preferred that all optional components are relatively resistant tooxidation by chlorine dioxide, since oxidation of composition componentsby chlorine dioxide will reduce the available chlorine dioxide foroxidation of chromagens on a tooth. “Relatively resistant” means that inthe time scale of preparing and using the chlorine dioxide-containingcomposition, the function of the optional component is not diminished,and the composition retains tooth whitening efficacy and remainssubstantially non-cytotoxic, substantially non-irritating, and does notsubstantially damage hard tooth tissue.

II. Method of Use

The method can be practiced by contacting a tooth surface with asubstantially non-cytotoxic composition comprising a bleaching agent inan efficacious amount. Advantageously, no pre-treatment of the gums isrequired, because the composition is substantially non-irritating andsubstantially non-cytotoxic. The duration of contact with the tooth toachieve a measurable degree of tooth whitening can be readily determinedby the skilled artisan in view of the teachings herein. Advantageously,even after prolonged contact, the composition does not substantiallydamage hard tooth tissue. Generally, duration of contact ranges fromseconds to minutes, preferably at least about 60 seconds, morepreferably at least about 1, 2, 3, 4, or 5 minutes, still morepreferably about 6, 7, 8, 9, or 10 minutes, yet more preferably about11, 12, 13, 14, or 15 minutes, though contact can range up to 16, 17,18, 19, or 20 minutes, and further up to 21, 22, 23, 24, 25, 26, 27, 28,29, or 30 minutes, and further up to about 35, 40, 45, 50, 55, or 60minutes or longer in some circumstances. In certain embodiments,duration of contact ranges between about 1 and about 60 minutes, morepreferably, from about 5 minutes to about 30 minutes, and morepreferably still, about 10 to about 20 minutes. In a preferredembodiment, duration of contact for a treatment is about 15 minutes.Treatment frequency is also readily determined by skilled artisan armedwith the present disclosure. Treatment may comprise one episode of toothcontact or more than one episode. Treatment episodes may be contiguous,separated in time (e.g., a few hours to a few days, a few days to a fewweeks, and also longer intervals including several months to a year ormore) or both.

Contact between the composition and the tooth surface can be achieved byany of a number of well-known methods in the art. The composition can bebrushed or spread onto the tooth surface. The composition can be presenton a flexible strip or patch that can be pressed against and molded tothe tooth surface. The composition can be used as an oral rinse;accordingly, the composition can be held in the mouth and allowed tocontact the teeth either statically, or with agitation within the mouthusing, for example, the tongue and cheeks. The composition can be placedin a dental tray, which is then placed in contact with the teeth. Suchtrays may be custom made or non-custom made. Numerous devices useful inpracticing the methods and using the compositions are disclosed in theart including, but not limited to U.S. Pat. Nos. 5,879,691; 6,551,579;6,682,721; 6,848,905; 6,896,518; 6,964,571; 7,004,756; 7,040,897; andU.S. Pat. Publication Nos. 2006/0223033; 2007/0298380; and 2008/0025925.

In some embodiments, the method can be practiced using a dental tray.The tray can be custom made. Methods of making custom-made trays arewell known in the art; see, for instance, U.S. Pat. Nos. 6,106,284 and6,425,759, and U.S. Pat. Publication Nos. 2006/0183080 and 2008/0041400.In brief, an impression tray can be filled with an impression material,such as alginate. The impression tray can then be positioned into themouth of the patient so as to create a negative impression of the teethin the impression material. After the negative impression has beenformed, the negative impression created in the impression tray can befilled with a soft casting material, such as dental stone, plaster orepoxy. The impression tray can be then inverted and mounted upon apre-formed mounting device, such as a dental cast tray or base. Afterthe casting material has had an opportunity to harden, the impressiontray is removed so that the casting material forms a positive dentalimpression on the mounting surface. Another method of preparing a customdental tray makes use of a “boil and bite” material, which is made outof a thermoformable plastic such as ethyl vinyl acetate (“EVA”) orpolyethylene. A customized tray can be created by heating thethermoformable plastic in boiling water causing it to melt at abiologically acceptable temperature, and then placing it directly overan individual's teeth where it cools and retains its new shape. Topractice a method, the substantially non-cytotoxic compositioncomprising a bleaching agent can be placed into the dental tray. Thetray is then positioned in the patient's mouth for the treatmentepisode.

It is also contemplated that administration of a chlorine dioxidecomposition may be made substantially non-cytotoxic by minimizing orprecluding contact of soft tissues with oxy-chlorine anions present inthe composition. Accordingly, as an example, devices comprising amicroporous barrier permeable to chlorine dioxide and substantiallynon-permeable to oxy-chlorine anions are envisioned. The chlorinedioxide composition may be completely or partially enclosed by such aselectively-permeable barrier. In some embodiments, the membrane can behydrophobic; the hydrophobic nature of the membrane prevents both anaqueous reaction medium and an aqueous recipient medium from passingthrough the membrane. Features to consider for the materials used forsuch a barrier include: hydrophobicity of the microporous material, poresize, thickness, and chemical stability towards the attack of chlorinedioxide, chlorine, chlorite, chlorate, chloride, acid and base. Ofcourse, for contact with soft tissues, the microporous barrier should besubstantially non-irritating and substantially non-cytotoxic,particularly in the time scale of typical use of the device. It isenvisioned that the chlorine dioxide composition utilized in such adevice need not be a thickened fluid, provided the device can be affixedto the tooth surface, and enable the chlorine dioxide that permeatesthrough the membrane to contact the tooth surface.

Materials useful as such barriers are known in the art and includeexpanded polytetrafluoroethylene (e.g., GORE-TEX) andpolyvinylidenefluoride (PVDF). See, for instance, U.S. Pat. No.4,683,039. The procedure for formation of a expandedpolytetrafluoroethylene is described in U.S. Pat. No. 3,953,566. Thematerial may be provided as a composite with supporting materials toprovide the structural strength required for use.

The pore sizes in the barrier may vary widely, depending on the desiredflow rate of the chlorine dioxide through the barrier. The pores shouldnot be so small as to prevent chlorine dioxide gas flow therethrough butalso should not be so large that liquid flow is permitted.

The porosity of the barrier may vary widely, also depending upon thedesired flow rate of chlorine dioxide through the barrier.Considerations of barrier strength also dictate the porosity chosen.Generally, the barrier porosity varies from about 50 to about 98%.

Also contemplated is the use of reactants for the formation of ClO₂embedded in a polymeric material that is permeable to ClO₂ butsubstantially non-permeable to oxy-chlorine anions. See e.g., U.S. Pat.No. 7,273,567.

As shown herein, a substantially non-cytotoxic composition comprisingchlorine dioxide as a bleaching agent has unexpected robust whiteningcapacity. Furthermore, a substantially non-cytotoxic compositioncomprising chlorine dioxide causes minimal damage to hard surfaces, suchas enamel and dentin, even during extended contact with an efficaciousamount on a tooth surface. In some embodiments, microhardness of enamelcontacted by a substantially non-cytotoxic composition is decreased lessthan about 15%, and preferably less than about 10%, less than about 8%and more preferably still, less than about 5%, relative to the enamelprior to contact. In some embodiments, enamel microhardness can bedecreased less than about 1% after a total treatment time of about seven(7) hours, relative to the enamel prior to contact. In some embodiments,microhardness of dentin contacted by a substantially non-cytotoxiccomposition is decreased less than about 15%, or less than about 10%,and or even less than about 8%, relative to the enamel prior to contact.In some embodiments, dentin microhardness can be decreased less thanabout 8% after a total treatment time of about seven (7) hours, relativeto the dentin prior to contact. Enamel and dentin surface roughness arealso not substantially increased by contact with a substantiallynon-cytotoxic composition. Preferably, surface roughness is increased byno more than about 20%, more preferably no more than about 15%, morepreferably no more than about 10%, and more preferably still, no morethan about 8%, relative to the surface roughness prior to contact.

Thus, as shown herein, a substantially non-cytotoxic chlorinedioxide-comprising composition advantageously provides a high degree oftooth whitening rapidly, with substantially no damage to hard toothtissue, no irritation of soft mucosal tissue, and no tooth sensitivity.This is in marked contrast to currently-available products based onhydrogen peroxide, which are cytotoxic and an irritant to soft tissuesand frequently cause tooth sensitivity and demineralization. Forinstance, professional tooth lightening hydrogen peroxide productsrequire steps prior to treatment to protect the gums during treatment.Both over-the-counter and professional hydrogen peroxide-based productsoften cause undesirable side effects, including pain, sensitivity andirritation of soft and/or hard tissues. Additionally, the use of anon-cytotoxic chlorine dioxide-comprising composition has minimaladverse effect on enamel and dentin microhardness and surface roughness.This combination of highly effective tooth lightening coupled withminimal unpleasant side effects is very desirable, and not achieved inprior art efforts. The method does not require the use of an externalactivator, such as a bleaching light or laser, and tooth whitening equalor superior to current over-the-counter bleaching materials may beobtained in less overall treatment time. In addition, tooth whiteningequal or superior to current professional bleaching materials can beobtained without cytotoxicity and irritation of soft tissue. Inparticular, the absence of soft tissue irritation advantageously permitsthe dental professional to proceed without a gum-protection step, whichcan take as long as about 90 minutes. Indeed, substantial contact withsoft oral tissue can be possible without irritation or cytotoxicity.“Substantial contact with soft oral tissue” as used herein refers tocontact that is more than contact with gum tissue proximal to a treatedtooth. Thus, substantial contact includes, but is not limited to,contact with gum, cheek mucosal and tongue tissue. Furthermore, theminimal effect on enamel and dentin microhardness reduces or eliminatesthe need for tooth sensitivity treatment and/or a remineralizationprocedure, both of which are often required with hydrogen peroxide-basedtooth lightening products. In addition, it has been suggested that theincreased surface roughness observed after hydrogen peroxide bleachingprovides an increased surface, which may facilitate early restainingafter whitening treatment. The method advantageously does not increasesurface roughness. Accordingly, it is contemplated that the method willalso provide a reduced rate and/or extent of rebound after whitening.

Chlorine dioxide slowly decays over time. Thus, when the method can bepracticed using a composition comprising chlorine dioxide, to maximizethe tooth whitening potency of the composition and to assurenon-cytotoxicity, the composition can be prepared immediately before useor is prepared in situ, as described elsewhere herein. Preparation canbe accomplished by methods described in commonly-assigned U.S.provisional application No. 61/150,685, filed Feb. 6, 2009, entitled“Non-Cytotoxic Chlorine Dioxide Fluids.”

In a preferred embodiment, a particulate precursor of chlorine dioxideis present in a first dispenser, such as a syringe, and a thickenercomponent in an aqueous medium is present in a second dispenser. Theaqueous thickened fluid in the second dispenser can be added directly tothe particulate mixture in the first dispenser, the combination allowedto react to produce ClO₂, and then mixed until homogeneous.Alternatively, an aqueous medium can be added to the particulateprecursor to prepare a substantially pure chlorine dioxide solution. Theappropriate amount of this solution can be then mixed with the aqueousthickener in the other dispenser. Both these embodiments areadvantageously practiced using syringes as the dispenser. In eitherembodiment, the two syringes can be connected to each other, and thecontents combined by dispensing the contents of one syringe into theother, then dispensing the mixture back into the other syringe until themixture is homogeneous. In another embodiment, the two dispensers arethe two barrels of a dual barrel syringe. Other devices to prepare anddispense the composition are described in commonly-assigned U.S.provisional application No. 61/150,685, filed Feb. 6, 2009, entitled“Non-Cytotoxic Chlorine Dioxide Fluids.”

III. Kits and Other Articles of Manufacture

Also provided herein is a kit comprising the composition, or theingredients therefore, and an instructional material, which describesusing the composition in a method of whitening a tooth. As used herein,an “instructional material,” includes a publication, a recording, adiagram, or any other medium of expression that can be used tocommunicate the usefulness of the composition and/or compound in a kit.The instructional material of the kit may, for example, be affixed to acontainer that contains the compound and/or composition or be shippedtogether with a container that contains the compound and/or composition.Alternatively, the instructional material may be shipped separately fromthe container with the intention that the recipient uses theinstructional material and the compound cooperatively. Delivery of theinstructional material may be, for example, by physical delivery of thepublication or other medium of expression communicating the usefulnessof the kit, or may alternatively be achieved by electronic transmission,for example by means of a computer, such as by electronic mail, ordownload from a website.

In an embodiment, the kit comprises two dispensers useful for preparingthe composition. One dispenser comprises a particulate precursor ofchlorine dioxide. The second dispenser comprises a thickener componentin an aqueous medium.

In another embodiment, the kit comprises a two-compartment container.One compartment comprises a particulate precursor of chlorine dioxide.The second compartment can comprise a thickener component in an aqueousmedium. Optionally, the container comprises a third compartment forcombining some or all of the contents of the two other compartments.

In some embodiments of the kit, the particulate precursor is ASEPTROLgranules, preferably ASEPTROL S-Tab2 granules. In some embodiments ofthe kit, the thickener component can be CMC. In preferred embodiments ofthe kit, the particulate precursor comprises ASEPTROL S-Tab2 granulesand the thickener component comprises CMC.

Optionally, the kit further comprises an applicator. By the term“applicator,” as the term is used herein, is meant any device including,but not limited to, a dental tray, a syringe, a pipette, a brush, a cup,and the like, suitable for contacting the tooth surface with thecomposition.

EXAMPLES

The compositions and methods of use are further described in detail byreference to the following experimental examples. These examples areprovided for purposes of illustration only, and are not intended to belimiting unless otherwise specified. Thus, the compositions, methods ofuse, and kits should in no way be construed as being limited to thefollowing examples, but rather, should be construed to encompass any andall variations which become evident as a result of the teaching providedherein.

Experimental Example 1 Cytotoxicity Analysis

To test the effects of chlorine dioxide on mammalian cells, thefollowing experiment was performed. Two series of samples comprisingdifferent amounts of chlorite anion were prepared. Examples 1-4 used asuper absorbent polyacrylate gel (labeled gel type “S”). Examples 5-8used a carboxymethylcellulose (CMC) gel (labeled gel type “C”).

ASEPTROL S-Tab2 granules were used in the gel compositions used in thisexperiment. The chemical composition of the granules is shown in Table1.

TABLE 1 Component % (wt/wt) Sodium chlorite 7% Dichloroisocyanuric acid,sodium salt 1% Sodium bisulfate 12% Sodium chloride 40% Magnesiumchloride 40%

Sodium chlorite (Aragonesas Energia of Spain) was technical grade,containing nominally 80% (0.8) by weight NaClO₂ and 20% inorganicstabilizer salts such as NaCl, NaOH, Na₂CO₃, and Na₂SO₄.Dichloroisocyanuric acid sodium salt (NaCl₂(CNO)₃.2H₂O) was obtainedfrom Oxychem as ACL-56.

The tablets, from which granules were made, were prepared as essentiallyas described in Example 4 of U.S. Pat. No. 6,432,322, incorporatedherein by reference. In brief, each of the separate components of thegranules was dried. The appropriate quantities of the components weremixed together and the mixture was compacted into tablet form using ahydraulic table press. The thus-formed tablets were ground into granulesusing a mortar and pestle. The resultant granules were screened using a40 mesh US Standard screen; the −40 mesh size fraction was used in theexperiments.

ASEPTROL S-Tab2 tablets have a high degree of conversion of chloriteanions to ClO₂ (see Examples in U.S. Pat. No. 6,432,322). Typically, asolution made from such tables will contain about 10× as much ClO₂ asresidual chlorite anion. When contacted with water (liquid), the wateris absorbed into the pores of the tablet, where it forms a saturatedaqueous solution of the constituents. Such conditions (highconcentration of chlorite anion and low pH) are advantageous for thereaction of chlorite anion (ClO₂ ⁻) with acid or chlorine to producechlorine dioxide (ClO₂) by reactions:

5NaClO₂+4H⁺—>4ClO₂+NaCl+4Na⁺+2H₂O   Eq. 3

2NaClO₂+OCl⁻+H⁺—>2ClO₂+NaCl+NaOH   Eq. 4

Residual chlorite anion in solution can result from several sources. Asource of residual chlorite anion in solution is sodium chlorite, whichdissolves from the exterior surface of an ASEPTROL tablet (or granule)into the bulk solution. The conversion rate of chlorite anion to ClO₂ islow at the very dilute and generally neutral-pH conditions of the bulksolution, so any chlorite anion that dissolves from the exterior of atablet or granule will remain substantially unconverted and remain aschlorite anion in solution. As a result, anything that enhances surfacedissolution of sodium chlorite prior to its conversion to ClO₂ willresult in an increase in chlorite anion concentration in the resultantsolution or gel.

Each base gel (aqueous thickened fluid) was slightly different tocompensate for the different active ingredient concentrations in thefinal samples. The final concentration of thickener component in theprepared gel samples was the same within each series. Each sample wasmade in an about 30 gram amount. The base gels were prepared bycombining deionized water with the gelling agents (thickener component).To allow the gelling agents to become fully hydrated, the mixtures wereallowed to stand for several hours to overnight. The base gel mixtureswere then stirred to homogenize the base gel.

The samples were prepared by combining ASEPTROL granules with a base gelshortly before use. The exposure of the ASEPTROL material to ambienthumidity or water was minimized prior to use to avoid loss of potency.After ASEPTROL granules were added to the base gel, the samples weremixed for 30 seconds with a stainless steel or plastic spatula, cappedand left to stand at room temperature for 5 minutes. The samples werethen mixed a second time for 30 seconds to homogenize the sample.Prepared samples were tightly capped until time of testing. The sodiumchlorite granules and the prepared samples were protected from strong uvlights to limit uv-induced decomposition. Testing was begun no more than2 hours after the samples were prepared.

Chlorine dioxide concentration was assessed by pH 7 buffered titrationusing potassium iodide (KI) and sodium thiosulfate on other samples.Samples 1 and 5 had zero chlorine dioxide. Samples 2 and 6 had about 30ppm ClO₂. Samples 3 and 7 had about 40 ppm and samples 4 and 8 had about580 ppm ClO₂.

There is not an extremely accurate method for measuring directlychlorite anions in a thickened fluid composition. Thus, the maximumconcentration of chlorite anion possibly present in each prepared sampleis provided below. It is expected that the actual amount of chloriteanion is less the maximum, as the reactants are activated in thepresence of an aqueous medium and generate chlorine dioxide, thusconsuming chlorite anions. The maximum amount of chlorite anion possiblypresent in a sample was calculated using the following formula:

-   ((wt. S_tab2 granules×wt. fraction sodium chlorite in granules×wt.    fraction chlorite in sodium chlorite x nominal wt. fraction of    sodium chlorite)×1000)/total wt of final sample. The weight fraction    of sodium chlorite used in S-Tab2 granules is 0.07. The weight    fraction of chlorite in sodium chlorite is 0.74. The nominal weight    fraction of actual sodium chlorite in the sodium chlorite powder    (i.e., the purity of the sodium chlorite) used in the granules is    0.8. Thus, for instance, the calculation of the milligrams of    oxy-chlorine anion per gram of gel for Ex. 2 is:

((0.143 g.×0.07×0.74×0.8)×1000)/30 grams final sample.

The final formulation for the examples is shown in Tables 2 and 3.

TABLE 2 Component Sample 1 Sample 2 Sample 3 Sample 4 Sodium 1.4 1.4 1.41.4 polyacrylate¹ NaCl 1 1 1 0 Polyethylene oxide² 1.6 1.6 1.6 1.6Deionized water 26 25.9 25.6 25.6 S-Tab2 granules (−40 0 0.143 0.3571.43 mesh) Maximum Mg 0 0.2 0.5 2.0 chlorite per gram gel ¹LUQUASORB1010, BASF Corp ²POLYOX WSR N3000, Dow Chemical Corp.

TABLE 3 Component Sample 5 Sample 6 Sample 7 Sample 8 Sodium 0.75 0.750.73 0.73 carboxymethylcellulose (NaCMC)¹ Na₂HPO₄ 0 0 0 0.2 Deionizedwater 29.3 29.3 29.3 29.2 S-Tab2 granules 0 0.143 0.357 1.43 (−40 mesh)Maximum Mg chlorite 0 0.2 0.5 2.0 per g gel ¹Sigma Aldrich 419338

Each prepared sample was tested in accordance with USP <87>. The methodinvolves determining the biological reactivity of mammalian cellcultures following contact with a topical gel product using an agardiffusion test. The cells in this test are L929 mammalian (mouse)fibroblast cells cultured in serum-supplemented MEM (minimum essentialmedium). A cell monolayer of greater than 80% confluence was grown at37° C. in a humidified incubator for not less than 24 hours and thenoverlaid with agar. The agar layer serves as a “cushion” to protect thecells from mechanical damage, while allowing diffusion of leachablechemicals from the test specimen. Materials to be tested at applied to apiece of filter paper, which were then placed on the agar.

Specifically, a paper disk was dipped in sterile saline to saturate thedisk. The amount of saline absorbed is determined (disk is weighedbefore and after wetting). A quantity of test specimen was dispensedonto the surface of the wetted disk. The specimen aliquot can be keptwithin the boundaries of the disk but is not spread out over the entiredisk. The disk with the specimen aliquot was weighed again to assess theamount of sample on the disk. The disk was then placed on top of theagar overlay. Cultures are evaluated periodically over time for evidenceof cytotoxicity and are graded on a scale of 0 (no signs ofcytotoxicity) to 4 (severe cytotoxicity), as summarized in Table 4. Asample is deemed to meet the requirements of the test if none of thecell culture exposed to the sample shows greater than mild cytotoxicity(grade 2) after 48 hours of testing. A sample showing grade 3 or 4reactivity during the 48 hours is deemed cytotoxic.

TABLE 4 Grade Reactivity Description of Reactivity Zone 0 None Nodetectable zone around or under specimen 1 Slight Some malformed ordegenerated cells under specimen 2 Mild Zone limited to area underspecimen 3 Moderate Zone extends to 0.5 to 1.0 cm beyond specimen 4Severe Zone extends greater than 1.0 cm beyond specimen

The volume tested of each prepared example in this experimental examplewas about 0.1 cc. The results are shown in Table 5.

TABLE 5 Maximum Gel Mg chlorite Sample # Type per g gel Test result 1 S0 Pass 2 0.2 Pass 3 0.5 Fail 4 2.0 Fail 5 C 0 Pass 6 0.2 Pass 7 0.5 Fail8 2.0 Fail Positive control Fail Negative control Pass

Samples 1, 2, 5 and 6 met the criteria of USP biological reactivity invitro, indicating biocompatibility. Samples 3, 4, 7 and 8 did not meetthe requirements of the USP biological test in vitro. Thus, gels havinga maximum concentration of chlorite anion greater than about 0.2 mgchlorite anion/gram gel produced cytotoxic effect in this experiment.These data suggest that cytotoxicity is related in a dose-dependentmanner to the presence of chlorine dioxide, oxy-chlorine anions, or someother constituent(s) of S-TAB2 granules.

Experimental Example 2 Cytotoxicity Analysis

To confirm that cytotoxicity was induced by oxy-chlorine anions and notby other possibly noxious ingredients, the following experiment wasperformed.

A series of samples was prepared to test various ingredients orconditions for their role in inducing cytotoxicity. ASEPTROL S-Tab10tablets were used to prepare some of the samples in this experiment. Thechemical composition of the tablets is shown in Table 6. ASEPTROLS-Tab10 tablets were prepared essentially as described in Example 5 ofU.S. Pat. No. 6,432,322.

TABLE 6 Component % (wt/wt) Sodium chlorite 26% Dichloroisocyanuricacid, sodium salt 7% Sodium bisulfate 26% Sodium chloride 20% Magnesiumchloride 21%

All of the samples comprised NaCMC as the thickener component. Samples9, 16 and 17 were prepared using −40 mesh fraction granules preparedfrom ASEPTROL S-Tab10 tablets. Samples 10, 19 and 20 were prepared usingthe ingredients of ASEPTROL S-Tab 10 tablets in a non-granulated form.Specifically, the five ingredients were dried and mixed to form a powderhaving the composition shown in Table 5; the powder was not compactedand granulated. Thus, samples 9 and 10 have identical chemicalcomposition but are made with the solid component in a differentphysical form. Similarly, samples 16 and 19 have identical compositions,as do samples 17 and 20, and samples 17 and 20, have the same chemicalcomposition. Samples 11-14 were prepared using a powder having a subsetof the ingredients in the ASEPTROL tablets, wherein one or moreingredients was replaced (see second column of Table 7 for details).Sample 15 contained substantially pure ClO₂. Sample 18 was NaCMC alone.

Samples 9-14 and 16-20 were prepared as described in ExperimentalExample 1. In brief, the samples were prepared by combining the solidfraction (e.g., ASEPTROL granules) with a base gel shortly before use.The base gel was NaCMC that was allowed to hydrate. After the solidfraction was added to the base gel, the samples were mixed for 30seconds with a stainless steel or plastic spatula, capped, and left tostand at room temperature for 5 minutes. The samples were then mixed asecond time for 30 seconds to homogenize the sample. Prepared sampleswere tightly capped until time of testing. The sodium chlorite granulesand other solid mixture comprising sodium chlorite, and the preparedsamples were protected from strong uv lights to limit uv-induceddecomposition. Testing was begun no more than 2 hours after the sampleswere prepared.

Sample 15 was prepared using a base gel of hydrated NaCMC and asubstantially pure chlorine dioxide solution that was prepared on thesame day the sample was prepared and the test begun. The base gel wasprepared by adding 0.75 gm of sodium carboxymethylcellulose powder(Sigma-Aldrich, 700,000 mole. wt., typ.) to 19.2 gm of deionized water,allowing the mixture to stand in a covered jar for overnight, and mixingto homogenize the base gel. The substantially pure chlorine dioxidesolution was prepared as follows: Twelve (12) ASEPTROL S-Tab10 tablets(1.5 grams each) were placed into 1 liter of potable tap water,producing a deep yellow colored source solution of >1000 ppm chlorinedioxide. Air was bubbled into the bottom of the source solution at arate of about 1 liter per minute to strip chlorine dioxide from thesource solution into the air. The resultant chlorine dioxide-laden airwas then bubbled into the bottom of 1 liter of deionized water to form asolution of pure chlorine dioxide. Only ClO₂ and possibly some watervapor was transferred from the source to the product solution. All thesalt ingredients remained behind in the source solution. As a result,the product solution was a substantially pure solution of ClO₂. Bubblingwas ended when the yellow color of the source solution was nearly gone.A sample of the substantially pure chlorine dioxide solution wasanalyzed for chlorine dioxide concentration using a Hach Model 2010UV/Visible spectrophotometer; the substantially pure solution was foundto contain 700 ppm chlorine dioxide by weight. Ten (10) grams of the 700ppm pure chlorine dioxide solution was added to the base gel and mixedto produce a gel containing about 233 ppm chlorine dioxide andsubstantially no oxy-chlorine anions. As above, the NaClO₂-containingcomponents and the prepared samples were protected from strong uv lightsto limit uv-induced decomposition. All dry solid ingredients wereprotected from water exposure (e.g., ambient humidity) as well.

The samples were tested as described in Experimental Example 1, exceptsamples 17 and 20 were tested at an 0.04 cc dose, rather than an 0.1 ccdose. Testing was begun no more than 2 hours after the samples wereprepared.

The results are shown in Table 7.

TABLE 7 Maximum Mg chlorite Result per gram of USP Sample # final gel<87> 9 Prepared with ASEPTROL S-Tab10 0.5 Fail granules 10 Prepared withnon-granulated 0.5 Fail ingredients of ASEPTROL S-Tab10 11 NaDCCAreplaced with cyanuric acid 0.5 Fail 12 NaClO₂ replaced with NaCl 0 Pass13 NaDCCA removed 0.5 Fail 14 NaClO₂ replaced with NaCl, and 0 PassNaDCCA replaced with cyanuric acid 15 Prepared with pure ClO₂ (no other0.5 Pass salts) 16 Sample 9 prepared with 3x the water 0.17 Fail 17Sample 9, 0.04 cc dose on disk 0.5 Fail 18 NaCMC alone with no granules,salts 0 Pass or ClO₂ 19 Sample 10 prepared with 3x the water 0.17 Fail20 Sample 10, 0.04 cc dose on disk 0.5 Fail Positive control 0 FailNegative control 0 Pass

Samples 9-11, 13, 16, 17, 19 and 20 all failed to meet the criteria forUSP biological reactivity in vitro. Thus, mimicking the elution-typetest of USP <87> did not alter the results (compare samples 10 and 19,and samples 9 and 16). Reducing the dose did alter the results (comparesample 9 and 17, and samples 10 and 20). These data indicate thatneither the dose used in the test, nor the use of gel with 3× the waterplayed a role in the observed cytotoxicity.

The results for samples 9 and 10 indicate that the physical form of theASEPTROL component does not noticeably affect the cytotoxicity. Theresults for samples 11 and 13 indicate that the presence of achlorine-producing agent, NaDCCA, does not noticeably affect thecytotoxicity. This result suggests that the observed cytotoxicity doesnot result from chlorine.

Samples 12, 14, 15, and 18 met the criteria for USP biologicalreactivity in vitro, indicating biocompatibility. These data indicatethat the cytotoxicity is not caused by the gellent alone (Sample 18).The observation that Sample 15, which contained pure ClO₂ and no othersalts, did not cause cytopathic effect indicates that chlorine dioxideitself is not the cause of cytotoxicity observed in the samplescomprising ASEPTROL S-Tab10 granules.

The common feature of samples 12, 14, 15, and 18 is that none containschlorite anion. Thus, none of samples 12, 14 and 18 containsoxy-chlorine anions. It is formally possible that sample 15, comprisingpure ClO₂, may contain some oxy-chlorine anions due to the decompositionof ClO₂; however, the amount is insignificant.

In view of these results, it is concluded that oxy-chlorine anions arethe causative agent underlying the cytotoxicity observed in theseexperiments.

Experimental Example 3 Cytotoxicity Analysis

The data in Experimental Example 1 indicate that the cytotoxicity ofoxy-chlorine anions is dose dependent. Specifically, cytotoxicity wasnot observed in gels having a maximum of 0.2 mg chlorite anion per gramgel, whereas cytotoxicity was observed in gels having a maximum of 0.5mg chlorite anion/gr. This experiment was designed to further examinethe cytotoxicity of chlorite anions, using sodium chlorite solution,which permits an more accurate estimate of chlorite anion concentrationin the thickened fluid compositions tested. In addition, thecytotoxicity of a commercially-available over-the-counter,peroxide-based, tooth whitening product, containing 10% hydrogenperoxide as the bleaching agent was also assessed.

Sample 22-24 were prepared by combining an aqueous solution of sodiumchlorite with a base gel shortly before use. Thus, none of samples 22-25contained chlorine dioxide. These samples also did not contain an acidsource or a free halogen source. Samples 22-24 were prepared by mixingthe aqueous sodium chlorite solution with the base gel for 30 seconds,capping the sample and letting it stand at room temperature for 5minutes, the mixing for another 30 seconds. Sample 25 was similarlyprepared but using water instead of a sodium chlorite solution. None ofsamples 22-25 contained an acid source or a free halogen source.

Sample 26 is an over-the-counter (OTC) product that is a gel containing10% hydrogen peroxide; the gel material was used as present on thefoil-wrapped strip.

Sample 21 was prepared using a substantially pure chlorine dioxidesolution prepared by reacting ASEPTROL S-Tab10 tablets into water.Specifically, one 1.5 mg tablet was reacted in 200 mL H₂O. The resultingchlorine dioxide solution was not sparged. Chlorine dioxideconcentration of the solution was about 733 ppm, as assessed using aHach Model 2010 uv-vis spectrophotometer. Sample 21 thus had about 244ppm ClO₂, after dilution of 1 part solution with 2 parts of gel

The cytotoxicity results are shown in Table 8.

TABLE 8 Result of Sample # Gel Mg chlorite per gel USP <87> 21 CMC 0Pass (Made with ~700 ppm ClO₂ solution) 22 0.04 Pass 23 1.0 Fail 24 2.0Fail 25 0 Pass 26 unknown OTC product with 10% hydrogen Fail peroxidePositive control Fail Negative control Pass

The results for Samples 22-24 indicate that chlorite anion at elevatedconcentration is cytotoxic to human cells, confirming the conclusionsfrom Experimental Example 2. The result for Sample 21 indicates that ahigh chlorine dioxide concentration thickened fluid composition that isnon-cytotoxic can be prepared using substantially pure chlorine dioxidesolution prepared using ASEPTROL S-Tab10 tablets.

This data also shows that 10% H₂O₂ is cytotoxic (Sample 26) to mammaliancells. Indeed, the reactivity zone extended more than 1 cm beyond thegel specimen, suggesting severe cytotoxicity.

Experimental Example 4 Additional Cytotoxicity Studies

To further examine the relationship between cytotoxicity andoxy-chlorine anion concentration in a thickened fluid composition, thefollowing experiment was performed.

Samples 27-31 were prepared by combining an aqueous solution of sodiumchlorite (10 ml) with 20 g of a base gel (hydrated high viscosity NaCMC)shortly before use. The NaCMC was a USP grade CMC, obtained fromSpectrum Chemical (stock # CA194); a 1% aqueous solution has a viscosityof about 1500-3000 cp. The base gel was prepared using 0.85 g. of NaCMCper 30 g final composition in order to achieve rheology equivalent tothat for the CMC obtained from Sigma Aldrich. None of samples 27-30contained chlorine dioxide. Sample 27 was similarly prepared but usingwater instead of a sodium chlorite solution. Samples 26-30 were preparedby mixing the aqueous sodium chlorite solution (or water) with the basegel until homogenous.

Sample 31, having the same relative composition as Sample 6 and about 40ppm chlorine dioxide, was prepared using a two-syringe mixing method.One syringe contained −40 mesh ASEPTROL S-Tab2 granules (p.048 g). Thesecond syringe contained the base gel (10 grams). The contents of thetwo syringes were combined as follows. The syringe containing thegranules was held with the tip pointing up. The outlet plug was removedand a nylon connector was attached. The other end of the nylon connectorwas attached to the outlet of the syringe containing the base gel. Theplunger of the gel syringe was slowly depressed, expelling the gel intothe granules. The gel-and-granules mixture was then allowed to sit for 5minutes to activate the granules thereby generating chlorine dioxide;the syringes remained connected during this period. After 5 minutes, thesyringe plungers were alternately depressed at a brisk rate to move themixture back and forth between the two syringe bodies at least 15 times,or until the sample was homogenous in color. The gel was then ready foruse the agar diffusion test of USP <87>.

The results of the cytotoxicity testing are shown in Table 9.

TABLE 9 Sample # Gel Mg chlorite per gel Result of USP <87> 27 CMC 0Pass 28 0.1 Pass 29 0.2 Fail 30 0.4 Fail 31 0.2* Pass Positive controlFail Negative control Pass *maximum amount of chlorite anion possiblypresent; calculated as described in Experimental Example 1

These data further support the discovery that chlorite anion iscytotoxic to human cells in a dose-dependent relationship. Sample 29,which contains 0.2 mg chlorite per gram final composition, failed thetest, whereas Sample 28, which contains 0.1 mg chlorite anion per gramdid not fail. This suggests that chlorine dioxide compositions havingless than 0.2 mg chlorite anion per gram composition are not cytotoxicto human cells. This outcome also supports the expectation that chloriteanions present in gels made with ASEPTROL granules or powders isconsumed in the generation of chlorine dioxide. Specifically, gelsprepared using ASEPTROL granules or powder and having a maximum possibleamount of 0.2 mg chlorite anion per gram final composition were found tobe non-cytotoxic. Thus, the apparent concentration of chlorite anions inthese gels is estimated to be less than 0.2 mg chlorite per gram.

Experimental Example 5 Tooth Whitening

Tooth whitening efficacy of a chlorine dioxide-containing CMC gel wasassessed in the following experiment. The composition of the gel wassubstantially identical to that of Sample 6 in Table 3, having a maximumpossible chlorite anion concentration of about 0.2 mg/gram final gelcomposition and about 40 ppm ClO₂. The results in Experimental Example 1revealed that this composition is not substantially cytotoxic.

The materials and methods used in this experiment are now described.

Color assessment: Two methods were used to assess tooth shade: 1) visualassessment by Vita Classical Shade Guide; and 2) spectrophotometry.Digital imaging and digital image analysis was also performed to measurecolor of tooth images.

Visual assessment by Vita Classical Shade Guide: Initial baseline shadeand subsequent shade change was assessed by direct comparison to astandard Vita shade guide. The Vita shade guide is arranged in thefollowing order (as recommended by the manufacturer) for valueassessment:

-   B1*A1*B2*D2*A2*C1*C2*D4*A3*D3*B3*A3.5*B4*C3*A4*C4, where B1 is the    brightest and C4 is the darkest. Two investigators determined the    closest shade match by visualizing the subject tooth against a    standardized black background, under controlled, standardized    fluorescent light conditions. In selected whitening experiments,    background light conditions were standardized using a hand-held LED    illuminating device at a fixed distance from the test specimen. The    agreement level between operators in shade selection was greater    than 80%. Disagreements in shade selection were never greater than 1    shade value unit (SVU).

Spectrophotometry: A clinical spectrophotometer, Vita EasyShade®(Vident, Brea, Calif.) was used to obtain electronic, quantitative dataabout shade measurement and specific color measurement parameters basedon the CIELAB L*a*b* color space. In this 3D color space system, “L” isthe lightness of an object (ranging from black to white) and is the onlydimension of color that may exist by itself; “a” is a measure of rednessor green-ness; and “b” is a measure of yellowness or blueness. Thedevice uses a D65 illuminant with a color temperature (in Kelvin) of6500 degrees. At the conclusion of any whitening treatment, ΔL, Δa, andΔb values were determined and recorded.

Digital images: Digital images of the teeth were taken using an SLRdigital camera/microscope (Olympus DPII Digital Camera/Microscope withOptiva Zoom 100 lens attachment). All images were obtained with standardmanually-entered settings. Approximate fixed lighting of flash apparatuswas configured to provide optimal, standardized imaging conditions.Samples were indexed or oriented repeatedly in a fixed orientation toinsure reproducible image alignment.

Naturally-stained teeth: Human teeth having an intrinsic internalstaining of D4 or lower (i.e., D4 through C4) were used asnaturally-stained teeth. Teeth were sectioned then prepare as describedbelow.

Tea-stained teeth: Human teeth with an intrinsic shade value greaterthan D4 (i.e., B1 through C2) were subjected to tea-based artificialstaining solution as follows. After sectioning the teeth, the exposeddentin surfaces were polished with silicon carbide paper. The dentinsurface was then etched with 37% phosphoric acid etching gel for 20-25seconds, rinsed with water for 30 seconds, and blotted to a moist dentincondition. The teeth were then subjected to continuous staining cycles(by immersion in concentrated tea staining solutions) until the stainintensity appeared unchanged on visual inspection (usually in the Vitashade range of C4-A4).

Tea-stained and naturally-stained teeth were prepared for treatment asfollows. The exposed dentin surfaces of the teeth were coated with threeseparate coats of clear nail polish; each coat was allowed to dry for atleast one hour before the next coat was applied. Teeth were then placedin tap water for at least 24 hours prior to testing. Prior to initiationof treatment, baseline tooth segment shade was assessed by qualitativeand quantitative color assessments. The teeth were mounted inmesio-distal orientation on a glass microscope slide. During treatmentsand between treatments, the teeth were stored in 100% humidity in aplastic bag. During the whitening assay, teeth were removed from theplastic bag, rinsed thoroughly to remove treatment or control whiteningagent, then subjected to qualitative and quantitative color assessment.

Non-cytotoxic ClO₂ gel: The non-cytotoxic ClO₂ aqueous gel materialtested was prepared substantially as described for Sample 6. In brief,thirty (30) grams of aqueous gel base was prepared by adding 0.85 gramsof high viscosity sodium

-   carboxymethylcellulose powder to 29.15 grams distilled water. The    mixture was allowed to hydrate for at least about 8 hours, then was    mixed to homogenize the base gel. To the about 30 grams aqueous    NaCMC base gel in a container, 0.143 grams ASEPTROL S-Tab2 granules    (−40 mesh) was added and mixed gently for 30 seconds. The container    was then tightly capped and the mixture allowed to stand for 5    minutes at room temperature. It was then remixed briefly and the    ClO₂ gel was then ready for use.

The concentrations of most of the constituents of the final gel can becalculated from mass balance or have been measured. The constituents aresummarized in Table 10. The remaining constituents are: about 40 ppmClO₂ by pH 7 titration and less than about 110 ppm un-reacted chloriteanion (ClO2).

TABLE 10 Chemical species Cyranuric NaCMC Water Na⁺ Mg⁺² Cl− SO₄ ⁻² acidConc., Weight % 2.8% 96.7% 0.10% 0.048% 0.26% 0.047% 0.002%

The prepared ClO₂ gel was a transparent to translucent, light yellow,viscous, pseudoplastic fluid. It had a yield point sufficient to retainits shape when applied in a 1-2 mm layer to teeth, but low enough to besubstantially removed from the tooth surface and soft oral tissue bywiping. The gel was soluble in additional water and can be removed fromthe mouth via rinsing or irrigation. Although ClO₂ is unstable and willslowly decompose over time, the concentration loss over 8 hours underproper storage conditions (kept in closed container, tightly capped orsealed, and minimize exposure to ultraviolet radiation) is less than20%.

Treatment: After mixing, the ClO₂ gel was drawn into a 60 ml plasticsyringe. The 60 ml syringe was used to hold the gel during the assay,and for dispensing gel into a 10 ml plastic syringe. The gel in the 10ml syringe was then dispensed directly onto the tooth section enamelsurfaces as follows. At time zero, about 1 to 1.5 ml gel was dispensedonto the enamel surface of each tooth segment attached to the glassmicroscope slide. The thickness of the resulting gel layer was about 1.5to 3.0 mm in depth. After dispensing the gel onto the tooth segments,the glass slide was placed in a 15 mm×8 mm plastic Ziplock® bag (SCJohnson Co., Racine, Wis.), containing small strips of wet paper towelwithin the bag to maintain 100% humidity in the bag. The paper towelstrips were positioned to eliminate any contact of the plastic bag withthe tooth and gel surfaces.

Upon conclusion of a testing interval (usually 15 minutes), the glassslide was removed from the plastic bag, and the gel was carefullyremoved with an extra soft bristle toothbrush and a gentle stream ofrunning tap water. The tooth segments on the slide were then analyzedfor shade and color, during which time the glass slide was maintainedperiodically in a plastic bag at 100% humidity to avoid undue colorartifact resulting from dehydration.

The gel application procedure was repeated as designed until theexperiment was concluded. The tested tooth segments were stored on theglass microscope slide in a 100% humidity for later referenceobservation.

Naturally-stained and tea-stained human teeth were treated with the ClO₂gel for a total of one (1) hour (4-15 minute consecutive treatments). Asingle batch of ClO₂ gel was used for these consecutive treatments. Forcomparison, other tea-stained teeth were treated with anover-the-counter whitening product containing 10% hydrogen. Treatmentwith the OTC product consisted of 30 minute treatments, in accordancewith the manufacturer's directions. At the end of a treatment, theresidual OTC product left on the tooth after removal of the strip wasremoved from the tooth using a soft bristle toothbrush and a gentlestream of running water. For the multiple day treatments (e.g., 7, 10and 14 days) using the OTC product, typically one 30 minute treatmentoccurred in the morning and the second 30 minute treatment occurred inthe evening.

The Vita Shade values for individual teeth at baseline and after 4×15minute treatments (total of 60 minutes) with ClO₂ gel are tabulated inTables 11 and 12. One of the six tea-stained teeth and one of the sixnaturally-stained teeth each achieved B1 as a result of treatment withClO₂ gel.

TABLE 11 Tea-stained teeth Specimen Baseline shade Post-treatment shadeSVUs T1 A4 C1 9.0 T2 C2 B1 6.0 T3 C4 A3 7.0 T4 A3 A2 9.0 T5 D3 A2 5.0 T6A4 D2 11.0

TABLE 12 Naturally-stained teeth Specimen Baseline shade Post-treatmentshade SVUs N1 A3-D3 D2-A2 >4.0 N2 A3.5-B4 B2 >9.0 N3 A3 B2-A1 >6.0 N4 A3B2-A1 >6.0 N5 A3 B1 8.0 N6 B4 A2 8.0

As shown in FIG. 1, after 30 minutes (2×15 minute treatments) of thenon-cytotoxic ClO₂-containing gel on tea-stained teeth (n=6), the totalshade change was well over 5 Shade Value Units-Vita (SVUs). Fornaturally-stained teeth (n=6), the total shade change was over 6 SVUs.After 45 minutes of treatment (3×15 minutes), the total shade change fornaturally-stained teeth was about 7 SVUs. After one (1) hour (4×15minute) of treatment of tea-stained teeth with the ASEPTROL gel, themean total shade change was 7.83 SVUs. The total shade change fornaturally-stained teeth treated with non-cytotoxic ClO₂ gel was aboutthe same.

The non-cytotoxic ClO₂-containing gel achieved marked lightening ofnaturally-stained teeth after the first 15 minute treatment, andcontinued lightening with continued treatment. In contrast, treatment ofnaturally-stained teeth with the OTC composition containing 10% hydrogenperoxide showed a much lower degree of lightening. There was a modestimprovement (1 SVU) observed after the first 30 minute treatment and nosignificant change after the second 30 minute treatment. Following theOTC product manufacturer's recommended 2×30 minute daily treatments forseven (7) days (total treatment time of 7 hours) resulted in anoticeable improvement (total shade change of 4.9 SVUs); however, theimprovement in tooth whitening was markedly less compared to the ClO₂composition after 4×15 minute consecutive treatments (total treatmenttime of 1 hour). Ten (10) days of treatment (2×30 min per day; totaltreatment time of 10 hours) with the OTC product yielded a total shadechange of 6.1 SVUs. Fourteen (14) days of treatment (total treatmenttime of 14 hours) with the OTC composition containing 10% hydrogenperoxide was needed to result in a total shade change comparable to whatwas achieved with non-cytotoxic ClO₂ gel in one hour of treatment. Thus,the ClO₂-containing gel provided a greater degree of lightening in asignificantly shorter time compared to the hydrogen peroxide-basedcomposition. Furthermore, the ClO₂-containing gel formulation isnon-cytotoxic, whereas the 10% hydrogen-peroxide formulation iscytotoxic (see Experimental Example 3).

The average ΔL value for tea-stained teeth treated for a total of onehour with ClO₂-containing gel was 9.3; the range of values for the 6specimens was 0.8 to 22 ΔL units. The average ΔL value fornaturally-stained teeth treated for a total of one hour withClO₂-containing gel was 8.07. The average change in lightness for teethtreated for a total treatment time of 7 hours with the OTC compositioncontaining 10% hydrogen peroxide was 6.32 ΔL units.

Thus, non-cytotoxic ClO₂-containing gel was extremely effective inbleaching tea-stained teeth and naturally-stained teeth. Notableimprovement in lightening was detected after the first 15 minutetreatment. The degree of lightening achieved after only two-15 minutestreatments with ClO₂-containing gel required 7 days of 2×30 minute dailytreatments with an OTC whitening product containing 10% hydrogenperoxide to achieve. The degree of lightening achieved after four-15minute treatments with ClO₂-containing gel required over 10 days oftreatment with the OTC composition containing 10% hydrogen peroxide toachieve.

Experimental Example 6 Efficacy of ClO₂-Containing Gel vs 36% HydrogenPeroxide

This experiment was designed to compare the tooth whitening efficacy ofa non-cytotoxic ClO₂-containing gel with a professional chair-sidewhitening gel containing 36% hydrogen peroxide. This is the highestconcentration of hydrogen peroxide currently in use in professionalchair-side products. The ClO₂-containing gel was prepared as describedin Example 5 and the treatment procedure was the same as in Example 5.

The results are summarized in FIG. 2. After 45 minutes of treatment, thewhitening efficacy of the non-cytotoxic ClO₂-containing gel (˜40 ppmClO₂) approached that of the professional gel. As is well known in theart, gels containing 36% hydrogen peroxide are highly irritating to softtissue in the oral cavity. Thus, achieving comparable tooth whiteningefficacy in the absence of soft tissue irritation is highly desirableand not possible with prior art products.

There are a variety of professional tooth whitening products on themarket which are used by dental professionals. Like the over-the-counterproducts, the professional products are peroxide based. Data for theefficacy of these products is available in the literature (see forinstance Operative Dentistry, 2007, 32-4: 322-327). The literaturevalues suggest that a non-cytotoxic ClO₂-containing gel, a much milderbleaching agent than peroxide, approaches the efficacy of manyperoxide-based professional products and, in some cases, may exceed theefficacy of peroxide-based professional products.

Experimental Example 7 Microhardness of Enamel and Dentin

Hydrogen peroxide is known to adversely affect tooth hard tissues. Toothsensitivity is a common side effect of professional teeth whitenproducts and is believe to originate in morphological changes in theenamel and dentin induced by the high concentration of peroxide. Manyprofessional products recommend the use of sodium fluoride toremineralize the teeth and potassium nitrate to reduce toothsensitivity. To characterize the effect of a non-cytotoxicClO₂-containing gel on enamel and dentin, the microhardness androughness of enamel and dentin was assessed before and after contactwith the chlorine dioxide-containing gel.

The composition of the ClO₂-containing gel is identical to thecomposition in Experimental 5. For both enamel and dentin experimentsusing the ClO₂-containing gel and an over-the-counter product (OTC)containing 10% H₂O₂, total treatment time was 7 hours, consisting of14×30 minute treatments. Multiple batches of ClO₂-containing gel wereprepared and used in this experiment. No batch of ClO₂-containing gelwas used for more than 2 hours. The 7-hour total treatment time is thesame as the treatment time recommended for the OTC product. However,this total treatment time is in great excess of the time needed toachieve tooth whitening with the non-cytotoxic chlorine dioxidecomposition comparable to OTC or professional peroxide-based products(see Examples 5 and 6). For the professional tooth whitening gelcomprising 36% hydrogen peroxide, contact was limited to one hour. Thetooth specimens were stored in tap water prior to, during and aftertreatment.

Microhardness was assessed using a CSM Dynamic Microhardness tester at 2newtons load, 30 seconds load/30 second unloading. Five enamel specimensand five dentin specimens were tested for changes in microhardness. Eachspecimen served as its own control (pre-treatment compared topost-treatment). For enamel hardness, ten measurements per specimen weretaken pre-treatment and post-treatment. Thus, there were 50pre-treatment measurements and 50 post-treatment measurements. Fordentin hardness, five measurements per specimen were taken pre-treatmentand post-treatment, yielding 25 pre-treatment measurements and 25post-treatment measurements. Microhardness data was calculated asVickers hardness values. Statistical analysis consisted of ANOVA(one-way) using an alpha level of 0.05.

Results for enamel hardness (in Vickers hardness values) are shown inTable 13.

TABLE 13 Statistical Compo- significant sition Pre-treatmentPost-treatment p-value (p < 0.05) ClO₂-gel 498.89 ± 70.64  507.40 ±69.92 0.5090 No OTC 711.57 ± 114.56  722.84 ± 141.85 0.8474 No product36% H₂O₂ 538.56 ± 109.30 455.72 ± 36.62 0.000768 Yes

-   Neither the ClO₂-containing gel nor the 10% H₂O₂ product induced a    statistically significant change in enamel hardness. In contrast,    the professional product induced a statistically-significant    reduction (>15% reduction) in enamel hardness. Thus, non-cytotoxic    ClO₂-containing gel whitens teeth with an efficacy comparable to    that induced by professional peroxide gels but without adversely    affecting enamel hardness.

Results for dentin microhardness (in Vickers hardness values) are shownin Table 14.

TABLE 14 Statistical significant Composition Pre-treatmentPost-treatment p-value (p < 0.05) ClO₂-gel 94.96 ± 9.63 87.65 ± 6.690.0031 Yes OTC product  98.35 ± 15.14 88.71 ± 6.02 0.0118 Yes 36% H₂O₂101.50 ± 21.48  83.45 ± 11.97 0.002212 Yes

-   Regarding dentin microhardness, the ClO₂-containing gel induced a    minor (7.7%) reduction in dentin microhardness, which was    statistically significant. The OTC product (10% peroxide)    demonstrated a similar minor (9.8%) reduction in dentin    microhardness, which was also statistically significant. Notably,    the professional gel induced a dramatic (˜18%) reduction in dentin    after only one hour of total contact time.

Thus, non-cytotoxic composition comprising an efficacious amount ofchlorine dioxide for tooth whitening has no statistically significanteffect on enamel microhardness and only a minor effect on dentinmicrohardness. The effect on dentin microhardness is comparable to thatinduced by a commercially-available over-the-counter tooth whiteningproduct.

Experimental Example 8 Surface Roughness of Enamel and Dentin

It has been suggested that increased roughness results in an increasedsurface area that facilitates rebound in whitened teeth. To study theeffect of non-cytotoxic chlorine dioxide containing compositions onsurface roughness, a Surftest 1700 Profilometer was used to assesssurface roughness of enamel and dentin before and after treatment withvarious whitening compositions.

Four enamel specimens and four dentin specimens were tested. Eachspecimen served as its own control. Twelve measurements per specimenwere taken pre-treatment and another twelve per specimen were takenpost-treatment. Contact time for the ClO₂-containing gel was a total of2.5 hours, consisting of 4-15 minute treatments and 3-30 minutetreatments. Additional 30 minute treatments with ClO₂-containing gelwere performed until a total treatment time of 7 hours was achieved. Thespecimens were stored in tap water prior to, during and after treatment.The non-cytotoxic chlorine dioxide-containing composition is the same asthat used in Experimental Example 8. An over-the-counter (OTC) whiteningproduct containing 10% hydrogen peroxide was also tested, in 14 -30minute consecutive treatments. A professional tooth whitening gelcontaining 36% hydrogen peroxide was also tested. For the professionaltooth whitening gel, contact was limited to one hour. Statisticalanalysis consisted of ANOVA (one-way) using an alpha level of 0.05.

The profilometry data for the non-cytotoxic chlorine dioxide compositionand the OTC product containing 10% hydrogen peroxide are shown in Table15.

TABLE 15 R_(a) values ANOVA Pre- Post-treatment p-value treatment 2.5hrs 7 hrs 2.5 hrs 7 hrs Enamel OTC 0.037692 ± 0.00914  N/D 0.048742 ±0.0157  N/D 0.04674* ClO₂ 0.024975 ± 0.002445 0.021508 ± 0.000888 0.02295 ± 0.000666 0.005799* 0.048636* Dentin OTC 0.032053 ± 0.007332N/D 0.051742 ± 0.00882 N/D 0.0000053* ClO₂  0.03998 ± 0.005542 0.03775 ±0.00466 0.044608 ± 0.00392 0.296884  0.027545* N/D = not determined.*statistically significant

The average surface roughness, Ra, of enamel before treatment with thenon-cytotoxic chlorine dioxide composition was about 0.025 and 0.021after 2.5 hours of treatment, and about 0.023 after 7 hours oftreatment. Thus, no adverse effect by the ClO₂-containing gel wasdetected on enamel average surface roughness, even despite extendedduration of contact. Indeed, enamel average surface roughness wasactually smoother by about 13-14% after 2.5 hours of ClO₂-containing geltreatment in this experiment, indicating an enamel polishing effectunexpected in the absence of an abrasive. Furthermore, the effect ondentin average surface roughness after 2.5 hours of treatment wasstatistically insignificant. After 7 hours of treatment, the increase indentin average surface roughness was only about 8%. As previously noted,7 hours of treatment is in excess of the time need to achieve toothwhitening with this composition comparable to OTC or professionalperoxide-based products (see Examples 5 and 6). Thus, the non-cytotoxicchlorine dioxide composition can produce tooth whitening comparable toOTC or professional products without substantial damage to enamel ordentin surface roughness.

In contrast, OTC product-treated teeth after the recommended 7 hours ofcontact time showed an increase of enamel surface roughness of greaterthan about 29%. The increase in dentin surface roughness after 7 hourswas greater than 60%. After only 1 hour of treatment, the professionalperoxide product, containing 36% hydrogen peroxide, increased surfaceroughness of both enamel and dentin by about 203%.

Regarding effect on enamel, there is little evidence of surfacemorphology alteration in enamel is observed in the specimen (FIG. 3B)treated with non-cytotoxic ClO₂-containing gel for 7 hours, compared tothe non-treated control tooth (FIG. 3A). Indeed, the fine-finishingscratches (result of tooth specimen preparation) are still evident. Incontrast, the photograph of the specimen (FIG. 3C) treated with the 36%peroxide gel for one hour reveals significant areas of enamel alterationand possible erosion.

As shown in FIG. 4A, the dentin surface morphology of a control(untreated) tooth has some dentinal tubules exposed or revealed, withother dentinal tubules hidden by a dentin smear layer. Some smear plugsare evident within exposed tubules. The surface of the representativespecimen treated with an OTC product containing 10% H₂O₂ (FIG. 4B)reveals a greater number of dentinal tubules exposed, compared withuntreated control dentin surface. Exposed tubules appear somewhatenlarged compared to control surface, and many tubules appear openwithout the presence of occluding smear plugs. The representative toothspecimen (FIG. 4C) treated with non-cytotoxic ClO₂-containing gel has agreater number of dentinal tubules exposed or revealed, compared tocontrol surface; but fewer in number and smaller in dimension than thetubules present in the OTC-treated specimen. Exposed tubules are presentas narrow “slits” with limited openings; some dentinal tubules arehidden by an apparent smear layer; some smear plugs evident withinexposed tubules. Thus, the surface of the tooth treated withnon-cytotoxic ClO₂-containing gel more closely resembles the surface ofthe control tooth.

While not wishing to be bound by theory, it is believed that thealteration in dentin surface induced by the 10% hydrogen peroxide gel,and therefore expected for the higher concentration professionalproducts, may underlie at least in part the tooth sensitivity issuecommon in over-the-counter and professional peroxide whitening products.

Experimental Example 9 Clinical Trial

The purpose of this human subject feasibility study is to evaluate theefficacy of a chair side, 1 hour (4 separate 15 minute treatments),in-office application of a non-cytotoxic tooth whitening compositioncomprising about 40 ppm chlorine dioxide and high viscosity sodium CMCas the thickener component. The composition will comprise no more thanabout 0.2 mg per gram composition oxy-chlorine anions. Shade change andtooth sensitivity to the tooth whitening agent, as well as patientresponse to the treatment, will be evaluated. Fifteen subjects will beenrolled in a clinical trial. Subjects will receive a 1 hour (4 separate15-minute treatments), in-office treatment with the experimental toothwhitening agent in this pilot, single-arm, non-controlled, prospective,case-controlled study. All subjects will be monitored at baseline,immediately post-application of the in-office treatment, 72 hourspost-application, and one week post application of the in-officetreatment. Trained examiners using Vita (Vita Zahnfabrik) shade guidesand color transparencies will monitor color changes. The Vita shadeguide is one of the acceptable evaluation methods included in the ADAguidelines. The transparencies will be used as a record of colorchanges. Tooth sensitivity will be monitored using a standardized scalefor the patient to mark at baseline, immediately post in officetreatment, 72 hours post in office treatment and one week post in officetreatment.

Subjects will be selected on the basis of having maxillary anteriorteeth that are shade Vita A3 or darker, as judged by comparison with avalue-oriented Vita shade guide. Subjects must be 18-65 years old, havegood general health, and have good dental health and oral hygiene.Patients with active caries, periodontal disease, large anterior crownsor restorations, previously-bleached or tetracycline-stained teeth willbe excluded from the study.

Upon acceptance into the study, each patient will be examined by one ofthe clinicians. A baseline Vita shade will be determined by twoevaluators; the consensus shade of the six test maxillary anterior teethand the six control mandibular teeth will be recorded by the dentist. Adigital color transparency (Photomed S1 Pro Digital Clinical Camera—FujiBody; Sigma Lens; Nikon Flash) will be made at a 1:1 magnification. Thematching Vita shade tab will be included in the photograph.

An alginate impression of the maxillary arch will be made and poured indental stone. Custom whitening trays will be fabricated for each patientusing the material and design recommended by the manufacturer. Allsubjects will receive a prophylaxis before starting treatment and willbe asked to mark a standardized scale to rate baseline sensitivity.

The clinical trial has been initiated, and preliminary data has beenobtained.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While the compositions, kits, and their methods of use have beendisclosed with reference to specific embodiments, it is apparent thatother embodiments and variations may be devised by others skilled in theart without departing from the true spirit and scope of the describedcompositions, kits and methods of use. The appended claims are intendedto be construed to include all such embodiments and equivalentvariations.

1. A method of whitening a tooth surface, the method comprising contacting the surface of a tooth with an efficacious amount of a substantially non-irritating composition, wherein the composition comprises a bleaching agent that whitens the contacted tooth surface, wherein the bleaching agent is chlorine dioxide.
 2. The method of claim 1, wherein the composition comprises about 5 to about 1000 ppm chlorine dioxide.
 3. The method of claim 1, wherein the composition comprises less than about 0.2 milligrams oxy-chlorine anion per gram composition.
 4. The method of claim 1, wherein the composition has a pH from about 4.5 to about
 11. 5. The method of claim 1, wherein the composition is a thickened fluid composition comprising a thickener component.
 6. The method of claim 5, wherein the thickener component is selected from the group consisting of natural hydrocolloids, semisynthetic hydrocolloids, synthetic hydrocolloids, and clay.
 7. The method of claim 1, wherein contacting the tooth with the non-irritating composition does not: substantially damage hard tooth tissue; substantially reduce enamel microhardness; substantially reduce dentin microhardness; cause tooth sensitivity and/or substantially increase surface roughness of at least one of enamel and dentin.
 8. The method of claim 7, wherein rate and/or extent of rebound is reduced compared to a peroxide bleaching agent.
 9. The method of claim 1, wherein the non-irritating composition contacts soft oral tissue.
 10. The method of claim 1, wherein the contacted tooth surface is whitened by at least about 3 shade value units.
 11. A method of whitening a tooth surface, the method comprising contacting the surface of a tooth with an efficacious amount of a composition that does not substantially damage hard tooth tissue, wherein the composition comprises a bleaching agent that whitens the contacted tooth surface, wherein the bleaching agent is chlorine dioxide.
 12. The method of claim 11, wherein the composition comprises about 5 to about 1000 ppm chlorine dioxide.
 13. The method of claim 11, wherein the composition comprises less than about 0.2 milligrams oxy-chlorine anion per gram composition.
 14. The method of claim 11, wherein the composition has a pH from about 4.5 to about
 11. 15. The method of claim 11, wherein the composition is a thickened fluid composition comprising a thickener component.
 16. The method of claim 15, wherein the thickener component is selected from the group consisting of natural hydrocolloids, semisynthetic hydrocolloids, synthetic hydrocolloids, and clay.
 17. The method of claim 11, wherein contacting the tooth with the composition does not: substantially reduce enamel microhardness; substantially reduce dentin microhardness; cause tooth sensitivity; and/or substantially increase surface roughness of at least one of enamel and dentin.
 18. The method of claim 17, wherein rate and/or extent of rebound is reduced compared to a peroxide bleaching agent.
 19. The method of claim 11, wherein the composition contacts soft oral tissue.
 20. The method of claim 1, wherein the contacted tooth surface is whitened by at least about 3 shade value units. 